diff --git "a/annotation_CSV/PMC5603727.csv" "b/annotation_CSV/PMC5603727.csv" new file mode 100644--- /dev/null +++ "b/annotation_CSV/PMC5603727.csv" @@ -0,0 +1,1354 @@ +anno_start anno_end anno_text entity_type sentence section +0 6 Roquin protein Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40 TITLE +34 42 hexaloop structure_element Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40 TITLE +60 66 3′-UTR structure_element Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40 TITLE +70 74 Ox40 protein Roquin recognizes a non-canonical hexaloop structure in the 3′-UTR of Ox40 TITLE +4 23 RNA-binding protein protein_type The RNA-binding protein Roquin is required to prevent autoimmunity. ABSTRACT +24 30 Roquin protein The RNA-binding protein Roquin is required to prevent autoimmunity. ABSTRACT +0 6 Roquin protein Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). ABSTRACT +99 122 costimulatory receptors protein_type Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). ABSTRACT +131 175 tumor necrosis factor receptor superfamily 4 protein Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). ABSTRACT +177 183 Tnfrs4 protein Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). ABSTRACT +187 191 Ox40 protein Roquin controls T-helper cell activation and differentiation by limiting the induced expression of costimulatory receptors such as tumor necrosis factor receptor superfamily 4 (Tnfrs4 or Ox40). ABSTRACT +2 28 constitutive decay element structure_element A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. ABSTRACT +30 33 CDE structure_element A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. ABSTRACT +57 72 triloop hairpin structure_element A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. ABSTRACT +114 120 Roquin protein A constitutive decay element (CDE) with a characteristic triloop hairpin was previously shown to be recognized by Roquin. ABSTRACT +12 24 SELEX assays experimental_method Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). ABSTRACT +45 66 U-rich hexaloop motif structure_element Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). ABSTRACT +84 109 alternative decay element structure_element Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). ABSTRACT +111 114 ADE structure_element Here we use SELEX assays to identify a novel U-rich hexaloop motif, representing an alternative decay element (ADE). ABSTRACT +0 18 Crystal structures evidence Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +23 26 NMR experimental_method Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +46 54 Roquin-1 protein Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +55 58 ROQ structure_element Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +77 86 hexaloops structure_element Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +94 99 SELEX experimental_method Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +108 111 ADE structure_element Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +122 125 ADE structure_element Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +154 158 Ox40 protein Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +159 165 3′-UTR structure_element Crystal structures and NMR data show that the Roquin-1 ROQ domain recognizes hexaloops in the SELEX-derived ADE and in an ADE-like variant present in the Ox40 3′-UTR with identical binding modes. ABSTRACT +10 13 ADE structure_element In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. ABSTRACT +23 26 CDE structure_element In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. ABSTRACT +70 74 Ox40 protein In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. ABSTRACT +78 84 Roquin protein In cells, ADE-like and CDE-like motifs cooperate in the repression of Ox40 by Roquin. ABSTRACT +45 66 hexaloop cis elements structure_element Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin. ABSTRACT +116 130 messenger RNAs chemical Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin. ABSTRACT +134 140 Roquin protein Our data reveal an unexpected recognition of hexaloop cis elements for the posttranscriptional regulation of target messenger RNAs by Roquin. ABSTRACT +1 7 Roquin protein Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. ABSTRACT +14 33 RNA-binding protein protein_type Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. ABSTRACT +105 109 Ox40 protein Roquin is an RNA-binding protein that prevents autoimmunity by limiting expression of receptors such as Ox40. ABSTRACT +30 33 RNA chemical Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. ABSTRACT +34 43 structure evidence Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. ABSTRACT +69 94 alternative decay element structure_element Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. ABSTRACT +139 145 Roquin protein Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. ABSTRACT +152 189 structural and biochemical techniques experimental_method Here, the authors identify an RNA structure that they describe as an alternative decay element, and they characterise its interaction with Roquin using structural and biochemical techniques. ABSTRACT +4 10 Roquin protein The Roquin protein is essential in T cells for the prevention of autoimmune disease. INTRO +56 64 Roquin-1 protein This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. INTRO +100 106 Met199 residue_name_number This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. INTRO +110 113 Arg residue_name This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. INTRO +202 206 mice taxonomy_domain This is evident from the so-called sanroque mutation in Roquin-1, a single amino acid exchange from Met199 to Arg that causes the development of systemic lupus erythematosus-like symptoms in homozygous mice. INTRO +4 9 Rc3h1 gene The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +14 19 Rc3h2 gene The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +40 48 Roquin-1 protein The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +53 61 Roquin-2 protein The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +74 85 vertebrates taxonomy_domain The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +158 162 mice taxonomy_domain The Rc3h1 and Rc3h2 genes, encoding for Roquin-1 and Roquin-2 proteins in vertebrates, respectively, have both been shown to be essential for the survival of mice, but apparently serve redundant functions in T cells. INTRO +54 65 deletion of experimental_method Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. INTRO +66 72 Roquin protein Consistently, CD4+ and CD8+ T cells with the combined deletion of Roquin-encoding genes are spontaneously activated and CD4+ T-helper cells preferentially differentiate into the Th1, Tfh or Th17 subsets. INTRO +0 8 Roquin-1 protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +81 105 co-stimulatory receptors protein_type Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +114 118 Icos protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +120 124 Ox40 protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +129 135 CTLA-4 protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +141 150 cytokines protein_type Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +159 177 interleukin (IL)-6 protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +182 204 tumour necrosis factor protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +212 233 transcription factors protein_type Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +242 246 IRF4 protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +248 253 IκBNS protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +258 262 IκBζ protein Roquin-1 was shown to negatively regulate expression of transcripts encoding for co-stimulatory receptors such as Icos, Ox40 and CTLA-4, for cytokines such as interleukin (IL)-6 and tumour necrosis factor or for transcription factors such as IRF4, IκBNS and IκBζ (refs). INTRO +26 56 structural and functional data evidence We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +64 72 Roquin-1 protein We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +73 76 ROQ structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +84 92 bound to protein_state We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +105 131 constitutive decay element structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +133 136 CDE structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +141 156 short stem loop structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +158 160 SL structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +192 195 RNA chemical We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +211 234 3′-untranslated regions structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +236 243 3′-UTRs structure_element We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +269 272 Tnf protein We have recently reported structural and functional data of the Roquin-1 ROQ domain bound to a canonical constitutive decay element (CDE), a short stem loop (SL) that acts as a cis-regulatory RNA element in the 3′-untranslated regions (3′-UTRs) of target genes such as Tnf (ref). INTRO +4 7 ROQ structure_element The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +25 51 extended winged helix fold structure_element The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +109 112 RNA chemical The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +171 174 Tnf protein The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +175 178 CDE structure_element The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +179 182 RNA chemical The ROQ domain adopts an extended winged helix fold that engages predominantly non-sequence-specific protein–RNA contacts and mainly recognizes the shape of the canonical Tnf CDE RNA. INTRO +4 19 structural data evidence The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +24 43 mutational analysis experimental_method The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +120 124 loop structure_element The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +129 133 stem structure_element The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +141 144 CDE structure_element The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +184 190 Roquin protein The structural data and mutational analysis indicated that a broader, extended range of sequence variations in both the loop and stem of the CDE element is recognized and regulated by Roquin. INTRO +43 60 crystal structure evidence At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +124 127 CDE structure_element At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +133 135 SL structure_element At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +152 171 second binding site site At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +178 197 double-stranded RNA chemical At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +199 204 dsRNA chemical At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +216 224 extended protein_state At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +225 228 ROQ structure_element At the same time, Tan et al. described the crystal structure and supporting functional data of a similar interaction with a CDE-like SL, and reported a second binding site for a double-stranded RNA (dsRNA) within an extended ROQ domain. INTRO +25 28 CDE structure_element The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported. INTRO +48 56 Roquin-2 protein The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported. INTRO +57 60 ROQ structure_element The structural basis for CDE recognition by the Roquin-2 ROQ domain has also been recently reported. INTRO +50 58 Roquin-1 protein We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). INTRO +63 71 Roquin-2 protein We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). INTRO +109 120 paracaspase protein_type We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). INTRO +121 126 MALT1 protein We found that the posttranscriptional activity of Roquin-1 and Roquin-2 is regulated through cleavage by the paracaspase MALT1 (refs). INTRO +9 14 MALT1 protein Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. INTRO +54 60 Roquin protein Enhanced MALT1-dependent cleavage and inactivation of Roquin, and thus less effective repression of target genes, result from increased strength of antigen recognition in T cells. INTRO +215 221 Roquin protein These findings suggest that dependent on the strength of cognate antigen recognition differential gene expression and cell fate decisions can be established in naive T cells by a graded cleavage and inactivation of Roquin. INTRO +51 67 binding affinity evidence In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +86 88 SL structure_element In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +105 112 3′-UTRs structure_element In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +123 128 mRNAs chemical In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +200 206 Roquin protein In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +221 223 SL structure_element In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +224 227 RNA chemical In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +262 268 Roquin protein In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +356 362 Roquin protein In addition to this mechanism, the composition and binding affinity of cis-regulatory SL elements in the 3′-UTRs of target mRNAs may determine the sensitivity to repression by the trans-acting factor Roquin. Defining the SL RNA structures that are recognized by Roquin is therefore essential for our understanding of posttranscriptional gene regulation by Roquin and its involvement in T-cell biology and T-cell-driven pathology. INTRO +88 91 RNA chemical Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +123 129 Roquin protein Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +159 174 U-rich hexaloop structure_element Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +175 177 SL structure_element Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +185 191 3′-UTR structure_element Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +195 199 Ox40 protein Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +200 208 bound to protein_state Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +213 221 Roquin-1 protein Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +222 225 ROQ structure_element Here we present structural and functional evidence for a greatly expanded repertoire of RNA elements that are regulated by Roquin as demonstrated with a novel U-rich hexaloop SL in the 3′-UTR of Ox40 bound to the Roquin-1 ROQ domain. INTRO +34 38 Ox40 protein We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +73 76 CDE structure_element We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +86 94 hexaloop structure_element We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +95 97 SL structure_element We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +98 102 RNAs chemical We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +128 185 Systematic Evolution of Ligands by Exponential Enrichment experimental_method We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +187 192 SELEX experimental_method We find an additive regulation of Ox40 gene expression based on both its CDE-like and hexaloop SL RNAs that we identified using Systematic Evolution of Ligands by Exponential Enrichment (SELEX) experiments. INTRO +4 26 X-ray crystallographic experimental_method Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +28 31 NMR experimental_method Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +33 64 biochemical and functional data evidence Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +79 98 mutational analysis experimental_method Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +121 128 triloop structure_element Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +133 141 hexaloop structure_element Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +142 144 SL structure_element Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +145 149 RNAs chemical Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +191 197 Roquin protein Our X-ray crystallographic, NMR, biochemical and functional data combined with mutational analysis demonstrate that both triloop and hexaloop SL RNAs contribute to the functional activity of Roquin in T cells. INTRO +0 5 SELEX experimental_method SELEX identifies novel RNA ligands of Roquin-1 RESULTS +23 26 RNA chemical SELEX identifies novel RNA ligands of Roquin-1 RESULTS +38 46 Roquin-1 protein SELEX identifies novel RNA ligands of Roquin-1 RESULTS +23 35 Roquin-bound protein_state We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. RESULTS +36 39 RNA chemical We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. RESULTS +83 88 SELEX experimental_method We set out to identify Roquin-bound RNA motifs in an unbiased manner by performing SELEX experiments. RESULTS +2 14 biotinylated protein_state A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. RESULTS +50 58 Roquin-1 protein A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. RESULTS +69 74 2–440 residue_range A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. RESULTS +95 99 RNAs chemical A biotinylated amino-terminal protein fragment of Roquin-1 (residues 2–440) was used to enrich RNAs from a library containing 47 random nucleotides over three sequential selection rounds. RESULTS +0 26 Next-generation sequencing experimental_method Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. RESULTS +28 31 NGS experimental_method Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. RESULTS +40 43 RNA chemical Next-generation sequencing (NGS) of the RNA before and after each selection round revealed that the starting pool represented about 99.6% unique reads in ∼4.2 × 106 sequences. RESULTS +0 22 Bioinformatic analysis experimental_method Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. RESULTS +26 29 NGS experimental_method Bioinformatic analysis of NGS data sets derived from the starting pool and enriched selection rounds revealed that the complexity was reduced to 78.6% unique reads in 3.7 × 106 sequences that were analysed after 3 rounds of selection and enrichment. RESULTS +4 7 NGS experimental_method For NGS data analysis, the COMPAS software (AptaIT, Munich, Germany) was applied. RESULTS +9 33 sequences were clustered experimental_method Enriched sequences were clustered into so-called patterns with highly homologous sequences. RESULTS +24 46 co-occurrence approach experimental_method Based on this so-called co-occurrence approach, patterns on the basis of frequent motifs were generated and were searched for prominent hexamer sequences (Supplementary Fig. 1a). RESULTS +14 27 5′-CGTTTT-3′, chemical We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). RESULTS +28 40 5′-GCGTTT-3′ chemical We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). RESULTS +42 54 5′-TGCGTT-3′ chemical We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). RESULTS +59 71 5′-GTTTTA-3′ chemical We identified 5′-CGTTTT-3′, 5′-GCGTTT-3′, 5′-TGCGTT-3′ and 5′-GTTTTA-3′ motifs that were also reconfirmed in an independent experiment (Supplementary Fig. 1a) and are located within highly similar sequences (Fig. 1a and Supplementary Fig. 1b). RESULTS +51 68 sanroque mutation mutant Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +85 88 RNA chemical Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +100 106 Roquin protein Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +149 154 SELEX experimental_method Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +188 196 Roquin-1 protein Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +221 226 M199R mutant Consistent with previous findings showing that the sanroque mutation does not impair RNA binding of Roquin, we found similarly enriched sequences in SELEX approaches using a corresponding Roquin-1 fragment harbouring the M199R mutation (Fig. 1a and Supplementary Fig. 1b). RESULTS +13 18 SELEX experimental_method Notably, our SELEX approach did not reveal the previously identified CDE sequence. RESULTS +69 72 CDE structure_element Notably, our SELEX approach did not reveal the previously identified CDE sequence. RESULTS +54 57 CDE structure_element We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. RESULTS +79 108 sequence clustering algorithm experimental_method We assume that the region of sequence identity in the CDE is too short for our sequence clustering algorithm. RESULTS +45 50 SELEX experimental_method Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). RESULTS +86 88 SL structure_element Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). RESULTS +134 138 loop structure_element Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). RESULTS +160 164 stem structure_element Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). RESULTS +187 191 stem structure_element Evaluation of the structural context for the SELEX-derived motif suggested a putative SL formation with six unpaired nucleotides in a loop followed by a 5–8 nt stem, with one base in the stem not being paired (Supplementary Fig. 1c). RESULTS +14 21 3′-UTRs structure_element Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +31 37 Roquin protein Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +65 83 5′-TGCGTTTTAGGA-3′ chemical Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +97 126 Motif-based sequence analysis experimental_method Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +128 132 MEME experimental_method Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +195 203 hexaloop structure_element Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +221 227 3′-UTR structure_element Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +231 235 Ox40 protein Searching the 3′-UTRs of known Roquin targets with the consensus 5′-TGCGTTTTAGGA-3′, obtained by Motif-based sequence analysis (MEME), revealed a homologous sequence with the potential to form a hexaloop structure in the 3′-UTR of Ox40 (Fig. 1b). RESULTS +57 64 3′-UTRs structure_element Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). RESULTS +79 84 mRNAs chemical Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). RESULTS +124 128 loop structure_element Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). RESULTS +143 147 stem structure_element Importantly, this motif is present across species in the 3′-UTRs of respective mRNAs and showed highest conservation in the loop and the upper stem sequences with a drop of conservation towards the boundaries of the motif (Fig. 1c,d). RESULTS +14 16 SL structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +35 40 SELEX experimental_method The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +84 109 alternative decay element structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +110 112 SL structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +114 117 ADE structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +118 120 SL structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +127 130 ADE structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +136 138 SL structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +168 171 CDE structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +177 179 SL structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +187 193 3′-UTR structure_element The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +197 201 Ox40 protein The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +202 206 mRNA chemical The predicted SL for the consensus SELEX-derived motif (from here on referred to as alternative decay element SL, ADE SL), the ADE-like SL, is positioned 5′ to another CDE-like SL in the 3′-UTR of Ox40 mRNA. RESULTS +5 8 CDE structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +14 16 SL structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +78 81 CDE structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +91 97 3′-UTR structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +101 104 Tnf protein This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +105 109 mRNA chemical This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +111 114 CDE structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +115 117 SL structure_element This CDE-like SL differs in the sequence of the upper stem from the canonical CDE from the 3′-UTR of Tnf mRNA (CDE SL) (Fig. 1d). RESULTS +0 3 NMR experimental_method NMR analysis of Roquin-bound SL RNAs RESULTS +16 28 Roquin-bound protein_state NMR analysis of Roquin-bound SL RNAs RESULTS +29 31 SL structure_element NMR analysis of Roquin-bound SL RNAs RESULTS +32 36 RNAs chemical NMR analysis of Roquin-bound SL RNAs RESULTS +8 11 NMR experimental_method We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. RESULTS +50 73 Roquin-1-binding motifs structure_element We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. RESULTS +87 92 SELEX experimental_method We used NMR to analyse the secondary structure of Roquin-1-binding motifs derived from SELEX. RESULTS +0 74 Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy experimental_method Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +76 81 NOESY experimental_method Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +83 86 NMR experimental_method Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +87 94 spectra evidence Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +102 106 free protein_state Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +107 110 RNA chemical Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +120 128 bound to protein_state Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +133 141 Roquin-1 protein Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +142 145 ROQ structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +175 178 ADE structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +179 181 SL structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +187 190 ADE structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +196 198 SL structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +206 212 3′-UTR structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +216 220 Ox40 protein Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +251 255 Ox40 protein Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +256 259 CDE structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +265 267 SL structure_element Imino one- and two-dimensional nuclear Overhauser enhancement spectroscopy (NOESY) NMR spectra of the free RNA and when bound to the Roquin-1 ROQ domain were recorded for the ADE SL, the ADE-like SL in the 3′-UTR of Ox40 and the previously identified Ox40 CDE-like SL (Fig. 2). RESULTS +4 7 NMR experimental_method The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +20 24 free protein_state The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +25 29 RNAs chemical The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +79 91 stem regions structure_element The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +99 116 hexa- and triloop structure_element The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +117 119 SL structure_element The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +177 181 RNAs chemical The NMR data of the free RNAs show that almost all predicted base pairs in the stem regions of the hexa- and triloop SL including the closing base pairs are formed in all three RNAs. RESULTS +62 65 G15 residue_name_number Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). RESULTS +75 77 G6 residue_name_number Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). RESULTS +86 89 ADE structure_element Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). RESULTS +90 92 SL structure_element Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). RESULTS +107 137 non-Watson–Crick G–G base pair bond_interaction Notably, we also found an unambiguous imino proton signal for G15, but not G6, in the ADE SL, indicating a non-Watson–Crick G–G base pair at this position (Fig. 2a). RESULTS +12 40 chemical shift perturbations evidence Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +42 46 CSPs evidence Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +104 107 ROQ structure_element Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +156 159 RNA chemical Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +195 198 ROQ structure_element Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +213 224 stem region structure_element Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +232 235 RNA chemical Significant chemical shift perturbations (CSPs) are observed for imino proton signals on binding to the ROQ domain, demonstrating that formation of protein–RNA complexes involves contacts of the ROQ domain to the stem region of the RNA ligands (Fig. 2, bases coloured red). RESULTS +53 76 Watson–Crick base pairs bond_interaction No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +98 101 ADE structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +102 104 SL structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +113 117 Ox40 protein No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +118 121 ADE structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +127 129 SL structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +130 134 RNAs chemical No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +155 156 A residue_name No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +157 158 U residue_name No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +182 187 bulge structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +195 199 Ox40 protein No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +200 203 ADE structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +209 211 SL structure_element No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +212 215 RNA chemical No imino correlations are observed for the predicted Watson–Crick base pairs at the bottom of the ADE SL and the Ox40 ADE-like SL RNAs, as well as for the A–U base pair flanking the bulge in the Ox40 ADE-like SL RNA (Fig. 2a,b), suggesting that these base pairs are dynamic. RESULTS +58 62 Ox40 protein In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes). RESULTS +63 66 CDE structure_element In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes). RESULTS +72 74 SL structure_element In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes). RESULTS +75 78 RNA chemical In contrast, all expected base pairs are observed for the Ox40 CDE-like SL RNA (Fig. 2c; see also Supplementary Notes). RESULTS +0 10 Structures evidence Structures of ROQ bound to ADE SL RNAs RESULTS +14 17 ROQ structure_element Structures of ROQ bound to ADE SL RNAs RESULTS +18 26 bound to protein_state Structures of ROQ bound to ADE SL RNAs RESULTS +27 30 ADE structure_element Structures of ROQ bound to ADE SL RNAs RESULTS +31 33 SL structure_element Structures of ROQ bound to ADE SL RNAs RESULTS +34 38 RNAs chemical Structures of ROQ bound to ADE SL RNAs RESULTS +17 23 Roquin protein To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +48 50 SL structure_element To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +78 83 SELEX experimental_method To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +97 103 solved experimental_method To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +104 122 crystal structures evidence To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +130 138 Roquin-1 protein To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +139 142 ROQ structure_element To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +150 158 bound to protein_state To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +179 182 RNA chemical To elucidate how Roquin can recognize the novel SL elements identified in the SELEX approach, we solved crystal structures of the Roquin-1 ROQ domain bound to these non-canonical RNA elements. RESULTS +4 14 structures evidence The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +18 21 ROQ structure_element The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +22 30 bound to protein_state The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +42 45 ADE structure_element The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +46 48 SL structure_element The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +91 95 Ox40 protein The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +96 99 ADE structure_element The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +105 107 SL structure_element The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +108 112 RNAs chemical The structures of ROQ bound to the 20-mer ADE SL (Supplementary Fig. 2a) and to the 22-mer Ox40 ADE-like SL RNAs (Fig. 3a) were refined to a resolution of 3.0 and 2.2 Å, respectively. RESULTS +8 18 structures evidence In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +23 26 RNA chemical In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +37 39 SL structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +56 64 hexaloop structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +123 126 ROQ structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +127 132 helix structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +133 135 α4 structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +163 165 β3 structure_element In both structures the RNA adopts an SL fold, where the hexaloop is located in the vicinity of the carboxy-terminal end of ROQ helix α4 and the N-terminal part of β3 (Fig. 3a,b and Supplementary Fig. 2a,b). RESULTS +4 9 dsRNA chemical The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +10 14 stem structure_element The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +76 79 Tnf protein The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +80 83 CDE structure_element The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +84 86 SL structure_element The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +87 90 RNA chemical The dsRNA stem is recognized in the same way as previously reported for the Tnf CDE SL RNA (Supplementary Fig. 2c–e). RESULTS +43 51 hexaloop structure_element As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +88 95 triloop structure_element As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +103 106 CDE structure_element As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +107 110 RNA chemical As may be expected, the recognition of the hexaloop is significantly different from the triloop in the CDE RNA (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +45 48 ADE structure_element Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +49 51 SL structure_element Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +56 59 ADE structure_element Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +65 67 SL structure_element Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +68 72 RNAs chemical Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +100 110 structures evidence Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +123 126 RNA chemical Interestingly, although the sequences of the ADE SL and ADE-like SL RNAs are different, the overall structures and protein–RNA contacts are virtually identical (Supplementary Fig. 2a,d,e). RESULTS +27 30 C19 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +31 36 bulge structure_element The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +42 58 non-Watson–Crick bond_interaction The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +59 61 G6 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +62 65 G15 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +66 75 base pair bond_interaction The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +99 101 U1 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +107 113 Trp184 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +118 124 Phe194 residue_name_number The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +132 135 ADE structure_element The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +141 143 SL structure_element The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +144 147 RNA chemical The only differences are a C19 bulge, the non-Watson–Crick G6–G15 base pair and the interaction of U1 with Trp184 and Phe194 in the ADE-like SL RNA (Supplementary Fig. 2a,e–g). RESULTS +82 91 structure evidence Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +99 103 Ox40 protein Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +104 107 ADE structure_element Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +113 115 SL structure_element Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +116 119 RNA chemical Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +151 155 Ox40 protein Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +156 162 3′-UTR structure_element Given their highly similar binding modes we focus the following discussion on the structure of the Ox40 ADE-like SL RNA, as it naturally exists in the Ox40 3′-UTR and was solved at higher resolution. RESULTS +47 67 double-stranded stem structure_element The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +75 79 Ox40 protein The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +80 83 ADE structure_element The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +89 91 SL structure_element The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +110 113 CDE structure_element The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +114 121 triloop structure_element The overall orientation and recognition of the double-stranded stem in the Ox40 ADE-like SL is similar to the CDE triloop. RESULTS +13 28 U-rich hexaloop structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +36 40 Ox40 protein Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +41 44 ADE structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +50 52 SL structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +53 56 RNA chemical Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +78 85 surface site Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +93 96 ROQ structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +135 138 CDE structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +139 146 triloop structure_element Notably, the U-rich hexaloop in the Ox40 ADE-like SL RNA binds to an extended surface on the ROQ domain that cannot be accessed by the CDE triloop (Fig. 3b,c) and includes a few pyrimidine-specific contacts. RESULTS +37 43 Phe255 residue_name_number For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). RESULTS +53 67 hydrogen bonds bond_interaction For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). RESULTS +102 105 U11 residue_name_number For example, the main chain atoms of Phe255 form two hydrogen bonds with the Watson–Crick face of the U11 base (Fig. 3d). RESULTS +16 25 structure evidence Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +33 36 Tnf protein Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +37 40 CDE structure_element Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +41 48 triloop structure_element Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +53 59 Tyr250 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +88 101 hydrogen bond bond_interaction Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +128 131 G12 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +183 191 hexaloop structure_element Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +232 238 Tyr250 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +271 274 U11 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +341 362 stacking interactions bond_interaction Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +372 375 U10 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +380 383 U11 residue_name_number Although in the structure of the Tnf CDE triloop the Tyr250 side chain engages only one hydrogen bond to the phosphate group of G12 (ref.), a number of contacts are observed with the hexaloop (Fig. 3d–f): the side chain hydroxyl of Tyr250 contacts the phosphate group of U11, while the aromatic ring is positioned by parallel and orthogonal stacking interactions with the U10 and U11 bases, on either side, respectively (Fig. 3e). RESULTS +17 23 Tyr250 residue_name_number In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). RESULTS +59 62 U13 residue_name_number In addition, the Tyr250 main-chain carbonyl interacts with U13 imino proton (Fig. 3d,e). RESULTS +0 6 Val257 residue_name_number Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +11 17 Lys259 residue_name_number Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +21 27 strand structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +28 30 β3 structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +58 61 UGU structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +62 69 triloop structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +77 80 Tnf protein Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +81 84 CDE structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +85 88 RNA chemical Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +139 147 hexaloop structure_element Val257 and Lys259 in strand β3 are too far to contact the UGU triloop in the Tnf CDE RNA, but mediate a number of contacts with the longer hexaloop. RESULTS +18 24 Lys259 residue_name_number The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +31 45 hydrogen bonds bond_interaction The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +75 78 U10 residue_name_number The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +83 86 U11 residue_name_number The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +133 139 Val257 residue_name_number The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +140 146 stacks bond_interaction The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +156 159 U11 residue_name_number The side chain of Lys259 forms hydrogen bonds with the phosphate groups of U10 and U11 (Fig. 3e,f) and the hydrophobic side chain of Val257 stacks with the U11 base (Fig. 3d,f). RESULTS +4 7 RNA chemical The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +8 12 stem structure_element The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +28 50 Watson–Crick base pair bond_interaction The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +52 54 C8 residue_name_number The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +55 58 G15 residue_name_number The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +66 74 hexaloop structure_element The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +75 77 SL structure_element The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +78 81 RNA chemical The RNA stem is closed by a Watson–Crick base pair (C8–G15 in the hexaloop SL RNA). RESULTS +19 21 G9 residue_name_number Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +27 33 stacks bond_interaction Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +131 134 G12 residue_name_number Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +142 145 CDE structure_element Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +146 153 triloop structure_element Interestingly, the G9 base stacks on top of this closing base pair and takes a position that is very similar to the purine base of G12 in the CDE triloop (Fig. 3b,c and Supplementary Fig. 2b). RESULTS +4 6 G9 residue_name_number The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. RESULTS +43 46 A14 residue_name_number The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. RESULTS +62 65 A14 residue_name_number The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. RESULTS +86 98 minor groove site The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. RESULTS +106 109 RNA chemical The G9 base does not form a base pair with A14 but rather the A14 base packs into the minor groove of the RNA duplex. RESULTS +38 58 stacking interaction bond_interaction This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +62 64 G9 residue_name_number This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +66 69 U10 residue_name_number This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +74 80 Tyr250 residue_name_number This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +88 91 ROQ structure_element This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +121 124 RNA chemical This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +125 129 stem structure_element This arrangement provides an extended stacking interaction of G9, U10 and Tyr250 in the ROQ domain at the 5′-side of the RNA stem (Fig. 3e). RESULTS +4 7 U11 residue_name_number The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). RESULTS +12 15 U13 residue_name_number The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). RESULTS +22 27 stack bond_interaction The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). RESULTS +67 70 ROQ structure_element The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). RESULTS +78 82 wing structure_element The U11 and U13 bases stack with each other in the vicinity of the ROQ domain wing (Fig. 3b,d,f). RESULTS +38 41 C12 residue_name_number This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. RESULTS +49 54 Ox-40 protein This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. RESULTS +55 58 ADE structure_element This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. RESULTS +64 66 SL structure_element This is possible by exposing the base C12 of the Ox-40 ADE-like SL towards the solvent, which accordingly does not show any contacts to the protein. RESULTS +27 30 CDE structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +31 33 SL structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +44 47 ADE structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +48 50 SL structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +55 58 ADE structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +64 66 SL structure_element In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +67 71 RNAs chemical In summary, similar to the CDE SL, both the ADE SL and ADE-like SL RNAs are recognized mainly by non-sequence-specific contacts. RESULTS +58 61 ROQ structure_element However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA. RESULTS +132 135 RNA chemical However, these involve an extended binding surface on the ROQ domain with a number of additional residues compared with the triloop RNA. RESULTS +0 3 NMR experimental_method NMR analysis of ROQ interactions with ADE SLs RESULTS +16 19 ROQ structure_element NMR analysis of ROQ interactions with ADE SLs RESULTS +38 41 ADE structure_element NMR analysis of ROQ interactions with ADE SLs RESULTS +42 45 SLs structure_element NMR analysis of ROQ interactions with ADE SLs RESULTS +13 29 NMR spectroscopy experimental_method We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +45 48 ROQ structure_element We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +71 74 ADE structure_element We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +84 87 CDE structure_element We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +93 95 SL structure_element We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +96 100 RNAs chemical We next used NMR spectroscopy to compare the ROQ domain interaction of ADE-like and CDE-like SL RNAs in solution. RESULTS +0 4 CSPs evidence CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +32 35 ROQ structure_element CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +61 65 Ox40 protein CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +66 69 ADE structure_element CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +75 77 SL structure_element CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +78 81 RNA chemical CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +150 153 CDE structure_element CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +154 157 SLs structure_element CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +167 173 Lys220 residue_name_number CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +175 181 Lys239 residue_name_number CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +182 188 Thr240 residue_name_number CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +193 199 Lys259 residue_name_number CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +200 206 Arg260 residue_name_number CSPs observed for amides in the ROQ domain on binding to the Ox40 ADE-like SL RNA (Fig. 4a,b) map to residues that also mediate key interactions with CDE SLs, such as Lys220, Lys239/Thr240 and Lys259/Arg260 (Fig. 4b). RESULTS +63 80 crystal structure evidence This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. RESULTS +131 146 binding surface site This is fully consistent with the interactions observed in the crystal structure (Supplementary Fig. 2c–e) and indicates a similar binding surface. RESULTS +32 47 CSP differences evidence However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +78 81 ROQ structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +92 96 Ox40 protein However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +97 100 ADE structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +106 108 SL structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +109 113 RNAs chemical However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +125 128 CDE structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +134 136 SL structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +137 140 RNA chemical However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +148 152 Ox40 protein However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +153 159 3′-UTR structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +181 184 Tnf protein However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +185 188 CDE structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +189 191 SL structure_element However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +192 195 RNA chemical However, there are also notable CSP differences when comparing binding of the ROQ domain to Ox40 ADE-like SL RNAs and to the CDE-like SL RNA in the Ox40 3′-UTR (Fig. 4c), or to the Tnf CDE SL RNA (Supplementary Fig. 3 and Supplementary Notes). RESULTS +13 19 Ser253 residue_name_number For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). RESULTS +64 68 Ox40 protein For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). RESULTS +69 72 ADE structure_element For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). RESULTS +78 80 SL structure_element For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). RESULTS +134 142 hexaloop structure_element For example, Ser253 is strongly affected only on binding to the Ox40 ADE-like SL (Fig. 4a,b) in line with tight interactions with the hexaloop (Fig. 3d). RESULTS +33 36 ROQ structure_element On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +61 64 ADE structure_element On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +78 81 ADE structure_element On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +87 89 SL structure_element On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +90 94 RNAs chemical On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +122 125 NMR experimental_method On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +126 133 spectra evidence On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +138 142 CSPs evidence On the other hand, comparison of ROQ domain binding with the ADE and with the ADE-like SL RNAs indicates almost identical NMR spectra and CSPs. RESULTS +73 76 RNA chemical This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e). RESULTS +96 99 ROQ structure_element This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e). RESULTS +118 122 RNAs chemical This is consistent with the very similar structural features and mode of RNA recognition of the ROQ domain with these RNAs (Supplementary Fig. 2a,d,e). RESULTS +0 19 Mutational analysis experimental_method Mutational analysis of the ROQ-ADE interaction RESULTS +27 30 ROQ structure_element Mutational analysis of the ROQ-ADE interaction RESULTS +31 34 ADE structure_element Mutational analysis of the ROQ-ADE interaction RESULTS +43 46 ROQ structure_element To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +105 142 electrophoretic mobility shift assays experimental_method To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +144 149 EMSAs experimental_method To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +172 175 ROQ structure_element To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +191 195 Ox40 protein To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +196 199 ADE structure_element To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +205 208 RNA chemical To examine the individual contributions of ROQ–hexaloop interactions for complex formation, we performed electrophoretic mobility shift assays (EMSAs) with variants of the ROQ domain and the Ox40 ADE-like RNA (Fig. 5a and Supplementary Fig. 4). RESULTS +33 42 wild-type protein_state Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). RESULTS +43 46 ROQ structure_element Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). RESULTS +68 76 affinity evidence Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). RESULTS +107 110 Tnf protein Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). RESULTS +111 114 CDE structure_element Analysis of the interaction with wild-type ROQ revealed an apparent affinity in a similar range as for the Tnf CDE (Fig. 5a and ) Table 2). RESULTS +110 127 crystal structure evidence We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). RESULTS +145 148 NMR experimental_method We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). RESULTS +149 153 CSPs evidence We next tested a set of mutants (Supplementary Fig. 4), which were designed based on contacts observed in the crystal structure (Fig. 3) and the NMR CSPs (Fig. 4a,b). RESULTS +31 42 ROQ-Tnf CDE complex_assembly In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +109 128 structural analysis experimental_method In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +147 153 Lys220 residue_name_number In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +155 161 Lys239 residue_name_number In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +163 169 Lys259 residue_name_number In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +174 180 Arg260 residue_name_number In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +222 233 replacement experimental_method In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +237 244 alanine residue_name In line with expectations from ROQ-Tnf CDE binding (see comparison in Supplementary Fig. 4) and based on our structural analysis, the key residues Lys220, Lys239, Lys259 and Arg260 strongly reduce or abolish binding after replacement by alanine. RESULTS +58 63 Y250A mutant We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +64 70 mutant protein_state We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +78 86 hexaloop structure_element We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +87 89 SL structure_element We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +90 93 RNA chemical We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +127 130 Tnf protein We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +131 134 CDE structure_element We also observe an almost complete loss of binding in the Y250A mutant to the hexaloop SL RNA, which had not been seen for the Tnf CDE previously (Fig. 5a). RESULTS +36 42 Tyr250 residue_name_number This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). RESULTS +68 76 hexaloop structure_element This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). RESULTS +106 127 stacking interactions bond_interaction This underlines the central role of Tyr250 for stabilization of the hexaloop structure and recognition by stacking interactions (Fig. 3b,e). RESULTS +0 8 Mutation experimental_method Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). RESULTS +12 18 Ser253 residue_name_number Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). RESULTS +38 42 CSPs evidence Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). RESULTS +50 64 NMR titrations experimental_method Mutation of Ser253, which shows large CSPs in the NMR titrations (Fig. 4a,b), does not significantly impair complex formation (Supplementary Fig. 4). RESULTS +10 31 chemical shift change evidence The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. RESULTS +113 116 U11 residue_name_number The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. RESULTS +121 124 U13 residue_name_number The large chemical shift change is probably caused by ring current effects induced by the close proximity of the U11 and U13 bases. RESULTS +11 17 mutant protein_state Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +25 29 wing structure_element Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +37 40 ROQ structure_element Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +49 54 S265Y mutant Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +152 155 Tnf protein Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +156 159 CDE structure_element Finally, a mutant in the wing of the ROQ domain (S265Y) does only slightly impair binding, as has been previously observed for the interaction with the Tnf CDE (Supplementary Fig. 4). RESULTS +20 31 replacement experimental_method This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. RESULTS +35 38 Tyr residue_name This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. RESULTS +68 71 RNA chemical This indicates that replacement by Tyr does not strongly affect the RNA interaction, and that some conformational variations are tolerated. RESULTS +10 29 mutational analysis experimental_method Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures. RESULTS +78 86 hexaloop structure_element Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures. RESULTS +103 121 crystal structures evidence Thus, the mutational analysis is fully consistent with the recognition of the hexaloop observed in our crystal structures. RESULTS +45 51 Tyr250 residue_name_number To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +55 63 Roquin-1 protein To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +67 71 Ox40 protein To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +72 76 mRNA chemical To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +117 149 retroviral reconstitution system experimental_method To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +153 159 Roquin protein To prove the contribution of the key residue Tyr250 in Roquin-1 to Ox40 mRNA recognition and regulation, we set up a retroviral reconstitution system in Roquin-deficient CD4+ T cells. RESULTS +27 32 Rc3h1 gene Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +33 36 2fl gene Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +37 39 fl gene Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +60 64 mice taxonomy_domain Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +83 91 Roquin-1 protein Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +92 93 2 protein Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +114 123 tamoxifen chemical Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +158 204 reverse tetracycline-controlled transactivator protein_type Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +205 209 rtTA protein Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +237 256 4-hydroxy tamoxifen chemical Isolated CD4+ T cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice harbouring floxed Roquin-1/2 encoding alleles, a tamoxifen-inducible Cre recombinase and the reverse tetracycline-controlled transactivator rtTA were treated in vitro with 4-hydroxy tamoxifen, to induce deletion. RESULTS +36 47 doxycycline chemical The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). RESULTS +93 101 Roquin-1 protein The cells were then transduced with doxycycline-inducible retroviral vectors to reconstitute Roquin-1 expression (Fig. 5b). RESULTS +13 19 Roquin protein Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). RESULTS +32 41 tamoxifen chemical Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). RESULTS +117 121 Ox40 protein Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). RESULTS +126 130 Icos protein Depletion of Roquin proteins on tamoxifen treatment (Supplementary Fig. 5a) strongly increased surface expression of Ox40 and Icos (Fig. 5c). RESULTS +44 67 costimulatory receptors protein_type This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). RESULTS +99 110 doxycycline chemical This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). RESULTS +139 147 Roquin-1 protein This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). RESULTS +148 150 WT protein_state This increase in surface expression of both costimulatory receptors was partially corrected by the doxycycline-induced reconstitution with Roquin-1 WT protein (Fig. 5c left panels). RESULTS +57 62 Y250A mutant Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +63 69 mutant protein_state Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +73 81 Roquin-1 protein Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +89 94 K220A mutant Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +96 101 K239A mutant Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +106 110 R260 mutant Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +111 117 mutant protein_state Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +149 152 CDE structure_element Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +153 155 SL structure_element Importantly, no effect was observed on expression of the Y250A mutant of Roquin-1 or the K220A, K239A and R260 mutant, which is strongly impaired in CDE SL interactions (Fig. 5c middle and right panels). RESULTS +45 59 overexpression experimental_method However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. RESULTS +81 87 Roquin protein However, it is also possible that continuous overexpression of targets following Roquin deletion induces a hyperactivated state in the T cells. RESULTS +127 131 Icos protein This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. RESULTS +136 140 Ox40 protein This hyperactivation, compared with the actual posttranscriptional derepression, may contribute even stronger to the increased Icos and Ox40 expression levels. RESULTS +11 38 structure–function analyses experimental_method Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +66 70 Y250 residue_name_number Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +96 102 Roquin protein Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +133 137 Ox40 protein Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +170 176 Roquin protein Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +193 197 Icos protein Hence, our structure–function analyses conclusively show that the Y250 residue is essential for Roquin interaction and regulation of Ox40, and potentially also for other Roquin targets such as Icos. RESULTS +63 67 Ox40 protein We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. RESULTS +68 71 ADE structure_element We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. RESULTS +77 79 SL structure_element We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. RESULTS +111 114 ROQ structure_element We also investigated the role of individual nucleotides in the Ox40 ADE-like SL for complex formation with the ROQ domain. RESULTS +141 161 co-crystal structure evidence We designed four mutants (Mut1–4, see Supplementary Fig. 6) that were expected to disrupt key interactions with the protein according to our co-crystal structure (Fig. 3d–f and Supplementary Fig. 2). RESULTS +0 3 NMR experimental_method NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +32 38 mutant protein_state NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +39 43 RNAs chemical NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +78 89 stem region structure_element NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +108 117 wild-type protein_state NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +118 121 ADE structure_element NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +127 129 SL structure_element NMR analysis confirmed that all mutant RNAs formed the same base pairs in the stem region, identical to the wild-type ADE-like SL (Fig. 2b and Supplementary Fig. 6). RESULTS +13 38 surface plasmon resonance experimental_method We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). RESULTS +64 86 dissociation constants evidence We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). RESULTS +95 98 ROQ structure_element We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). RESULTS +99 102 RNA chemical We next used surface plasmon resonance experiments to determine dissociation constants for the ROQ-RNA interaction (Table 2 and Supplementary Fig. 7). RESULTS +13 24 replacement experimental_method Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +30 32 C8 residue_name_number Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +33 36 G15 residue_name_number Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +58 59 A residue_name Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +60 61 U residue_name Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +63 68 Mut 4 mutant Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +87 95 affinity evidence Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +120 124 loop structure_element Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +137 141 A14C mutant Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +142 148 mutant protein_state Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +150 155 Mut 1 mutant Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +182 190 affinity evidence Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +222 247 surface plasmon resonance experimental_method Although the replacement of a C8–G15 closing base pair by A-U (Mut 4) only reduces the affinity threefold, reduction of loop size in the A14C mutant (Mut 1, see Table 2) reduces the affinity and binding is not detected by surface plasmon resonance. RESULTS +26 31 Mut 1 mutant As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). RESULTS +117 126 tetraloop structure_element As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). RESULTS +138 139 G residue_name As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). RESULTS +140 141 C residue_name As intended, the mutation Mut 1 allows the formation of an additional base pair and thus leads to the formation of a tetraloop with a new G-C closing base pair (Supplementary Fig. 6a). RESULTS +20 39 structural analysis experimental_method Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. RESULTS +80 88 hexaloop structure_element Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. RESULTS +140 143 ROQ structure_element Consistent with the structural analysis, we assume that this variant alters the hexaloop conformation and thus reduces the interaction with ROQ. RESULTS +14 35 stacking interactions bond_interaction Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +44 47 G15 residue_name_number Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +49 51 G9 residue_name_number Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +56 60 Y250 residue_name_number Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +68 71 G9C mutant Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +72 78 mutant protein_state Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +80 85 Mut 2 mutant Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +119 122 ROQ structure_element Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +130 132 SL structure_element Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +133 136 RNA chemical Disruption of stacking interactions between G15, G9 and Y250 in the G9C mutant (Mut 2) completely abolished binding of ROQ to the SL RNA (Table 2 and Supplementary Fig. 7). RESULTS +36 44 U11AU13G mutant No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +45 58 double mutant protein_state No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +60 65 Mut 3 mutant No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +153 156 U11 residue_name_number No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +161 164 U13 residue_name_number No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +172 180 hexaloop structure_element No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +186 189 ROQ structure_element No binding is also observed for the U11AU13G double mutant (Mut 3) (Table 2 and Supplementary Fig. 7), which abolishes specific interactions mediated by U11 and U13 in the hexaloop with ROQ (Fig. 3d). RESULTS +20 25 SELEX experimental_method Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +65 74 mutations experimental_method Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +78 87 conserved protein_state Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +88 99 nucleotides chemical Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +107 111 loop structure_element Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +151 154 ROQ structure_element Consistent with the SELEX consensus (Fig. 1b), all of the tested mutations of conserved nucleotides in the loop reduce or abolish the interaction with ROQ. RESULTS +19 27 affinity evidence Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +35 44 wild-type protein_state Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +45 48 Tnf protein Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +49 52 CDE structure_element Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +61 65 Ox40 protein Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +66 69 ADE structure_element Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +75 78 SLs structure_element Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +82 85 ROQ structure_element Interestingly, the affinity of the wild-type Tnf CDE and the Ox40 ADE-like SLs to ROQ are very similar (42 and 81 nM, respectively, Table 2 and Supplementary Fig. 7). RESULTS +0 6 Roquin protein Roquin binding to different SLs in the Ox40 3′-UTR RESULTS +28 31 SLs structure_element Roquin binding to different SLs in the Ox40 3′-UTR RESULTS +39 43 Ox40 protein Roquin binding to different SLs in the Ox40 3′-UTR RESULTS +44 50 3′-UTR structure_element Roquin binding to different SLs in the Ox40 3′-UTR RESULTS +28 36 Roquin-1 protein We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). RESULTS +48 51 CDE structure_element We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). RESULTS +70 76 3′-UTR structure_element We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). RESULTS +80 84 Ox40 protein We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). RESULTS +85 89 mRNA chemical We have recently shown that Roquin-1 binds to a CDE-like motif in the 3′-UTR of Ox40 mRNA (Figs 1d and 4c). RESULTS +60 63 CDE structure_element We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +77 80 ADE structure_element We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +86 88 SL structure_element We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +89 93 RNAs chemical We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +113 121 Roquin-1 protein We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +152 163 full-length protein_state We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +164 168 Ox40 protein We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +169 175 3′-UTR structure_element We therefore investigated whether the interactions with the CDE-like and the ADE-like SL RNAs both contribute to Roquin-1 binding in the context of the full-length Ox40 3′-UTR. RESULTS +4 22 binding affinities evidence The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +47 64 N-terminal domain structure_element The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +68 76 Roquin-1 protein The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +87 92 2–440 residue_range The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +127 130 ROQ structure_element The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +138 143 alone protein_state The binding affinities of either motif for the N-terminal domain of Roquin-1 (residues 2–440) (Supplementary Fig. 8a,b) or the ROQ domain alone are in a similar range (Table 2). RESULTS +4 26 dissociation constants evidence The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +35 38 ROQ structure_element The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +60 64 Ox40 protein The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +65 68 CDE structure_element The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +74 76 SL structure_element The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +85 88 ADE structure_element The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +94 96 SL structure_element The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +97 101 RNAs chemical The dissociation constants for the ROQ interaction with the Ox40 CDE-like SL and the ADE-like SL RNAs are 1,460 and 81 nM, respectively (Table 2). RESULTS +37 54 binding interface site This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +101 109 hexaloop structure_element This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +154 162 hexaloop structure_element This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +163 165 SL structure_element This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +166 169 RNA chemical This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +177 181 Ox40 protein This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +182 188 3′-UTR structure_element This is consistent with the extended binding interface and additional interactions observed with the hexaloop, and suggests a preferential binding to the hexaloop SL RNA in the Ox40 3′-UTR. RESULTS +38 44 3′-UTR structure_element We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +48 65 point mutagenesis experimental_method We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +97 108 stem region structure_element We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +142 144 SL structure_element We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +145 148 RNA chemical We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +161 168 mutated experimental_method We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +179 187 Roquin-1 protein We designed different variants of the 3′-UTR by point mutagenesis abrogating base pairing in the stem region, where none, individual, or both SL RNA motifs were mutated to impair Roquin-1 binding (Fig. 6a). RESULTS +6 10 RNAs chemical These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). RESULTS +31 36 EMSAs experimental_method These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). RESULTS +46 54 Roquin-1 protein These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). RESULTS +76 81 2–440 residue_range These RNAs were then tested in EMSAs with the Roquin-1 N terminus (residues 2–440) (Fig. 6b). RESULTS +0 16 Gel shift assays experimental_method Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. RESULTS +42 51 wild-type protein_state Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. RESULTS +52 58 3′-UTR structure_element Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. RESULTS +108 118 titrations experimental_method Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. RESULTS +165 168 RNA chemical Gel shift assays show that binding to the wild-type 3′-UTR construct leads to two distinct bands during the titrations, which should reflect binding to one and both RNA motifs, respectively. RESULTS +126 129 SLs structure_element Consistent with this, both bands are strongly reduced when mutations are introduced that interfere with the formation of both SLs. RESULTS +82 84 SL structure_element Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. RESULTS +85 88 RNA chemical Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. RESULTS +117 123 Roquin protein Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. RESULTS +178 187 wild-type protein_state Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. RESULTS +188 190 SL structure_element Notably, among these, the slower migrating band disappears when either of the two SL RNA motifs is altered to impair Roquin binding, indicating an interaction with the remaining wild-type SL. RESULTS +22 28 Roquin protein We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +53 55 SL structure_element We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +56 59 RNA chemical We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +89 100 full-length protein_state We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +101 105 Ox40 protein We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +106 112 3′-UTR structure_element We thus conclude that Roquin is able to bind to both SL RNA motifs in the context of the full-length Ox40 3′-UTR. RESULTS +14 18 Ox40 protein Regulation of Ox40 expression via two motifs in its 3′-UTR RESULTS +52 58 3′-UTR structure_element Regulation of Ox40 expression via two motifs in its 3′-UTR RESULTS +35 38 ADE structure_element To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +60 64 mRNA chemical To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +80 90 introduced experimental_method To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +91 95 Ox40 protein To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +96 100 mRNA chemical To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +121 128 altered protein_state To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +129 136 3′-UTRs structure_element To investigate the role of the new ADE-like motif in target mRNA regulation, we introduced Ox40 mRNA variants harbouring altered 3′-UTRs in cells. RESULTS +39 42 ADE structure_element Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +52 55 CDE structure_element Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +61 63 SL structure_element Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +64 68 RNAs chemical Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +76 82 3′-UTR structure_element Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +117 123 Roquin protein Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +167 171 Ox40 protein Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +256 259 RNA chemical Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +272 302 deletion and point mutagenesis experimental_method Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +303 313 abrogating protein_state Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +314 326 base pairing bond_interaction Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +334 345 stem region structure_element Considering the close proximity of the ADE-like and CDE-like SL RNAs in the 3′-UTR (Fig. 6a), which is essential for Roquin-mediated posttranscriptional regulation of Ox40 (ref.) we tested individual contributions and the functional cooperation of the two RNA elements by deletion and point mutagenesis abrogating base pairing in the stem region (Fig. 6a,c and Supplementary Fig. 8c). RESULTS +20 32 retroviruses taxonomy_domain Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +47 51 Ox40 protein Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +112 119 3′-UTRs structure_element Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +125 133 Roquin-1 protein Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +134 135 2 protein Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +146 151 mouse taxonomy_domain Specifically, using retroviruses we introduced Ox40 expression constructs placed under the control of different 3′-UTRs into Roquin-1/2-deficient mouse embryonic fibroblasts. RESULTS +0 11 Doxycycline chemical Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). RESULTS +67 75 Roquin-1 protein Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). RESULTS +162 172 lentiviral taxonomy_domain Doxycycline treatment of cells from this cell line enabled ectopic Roquin-1 and co-translational mCherry expression due to the stable integration of an inducible lentiviral vector (Supplementary Fig. 8c). RESULTS +18 22 Ox40 protein The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). RESULTS +49 60 doxycycline chemical The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). RESULTS +94 108 flow cytometry experimental_method The expression of Ox40 in cells with and without doxycycline treatment was then quantified by flow cytometry (Supplementary Fig. 8c). RESULTS +23 27 Ox40 protein Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +28 57 mean fluorescence intensities evidence Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +84 95 doxycycline chemical Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +157 161 Ox40 protein Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +173 180 without protein_state Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +181 187 3′-UTR structure_element Comparing the ratio of Ox40 mean fluorescence intensities in cells with and without doxycycline treatment normalized to the values from cells that expressed Ox40 constructs without 3′-UTR revealed a comparable importance of both structural elements (Fig. 6c). RESULTS +14 43 deletion or point mutagenesis experimental_method In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +104 110 3′-UTR structure_element In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +111 115 1–80 residue_range In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +120 130 double mut protein_state In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +144 150 Roquin protein In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +175 179 Ox40 protein In fact, only deletion or point mutagenesis of the sequences encoding both structures at the same time (3′-UTR 1–80 and double mut) neutralized Roquin-dependent repression of Ox40. RESULTS +24 33 mutations experimental_method In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +48 56 hexaloop structure_element In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +58 64 3′-UTR structure_element In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +65 70 1–120 residue_range In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +74 81 CDE mut mutant In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +90 93 CDE structure_element In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +99 106 triloop structure_element In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +107 113 intact protein_state In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +128 134 Roquin protein In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +214 225 full-length protein_state In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +226 232 3′-UTR structure_element In contrast, individual mutations that left the hexaloop (3′-UTR 1–120 or CDE mut) or the CDE-like triloop intact still enabled Roquin-dependent repression, which occurred in an attenuated manner compared with the full-length 3′-UTR (Fig. 6c). RESULTS +50 56 Roquin protein To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). RESULTS +72 78 3′-UTR structure_element To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). RESULTS +97 113 mRNA decay rates evidence To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). RESULTS +146 150 Ox40 protein To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). RESULTS +278 289 doxycycline chemical To further analyse the functional consequences of Roquin binding to the 3′-UTR, we also measured mRNA decay rates after introducing the different Ox40 constructs into HeLa tet-off cells that allow to turn off transcription from the tetracycline-repressed vectors by addition of doxycycline (Fig. 6d). RESULTS +0 38 Quantitative reverse transcriptase–PCR experimental_method Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +78 82 Ox40 protein Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +83 87 mRNA chemical Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +91 102 deletion of experimental_method Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +107 113 3′-UTR structure_element Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +115 118 CDS structure_element Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +119 123 t1/2 evidence Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +135 146 full-length protein_state Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +147 151 t1/2 evidence Quantitative reverse transcriptase–PCR revealed a strong stabilization of the Ox40 mRNA by deletion of the 3′-UTR (CDS t1/2=311 min vs full-length t1/2=96 min). RESULTS +43 60 combined mutation experimental_method A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +68 71 CDE structure_element A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +85 88 ADE structure_element A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +94 97 SLs structure_element A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +99 102 ADE structure_element A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +103 106 CDE structure_element A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +112 115 mut protein_state A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +116 120 t1/2 evidence A comparable stabilization was achieved by combined mutation of the CDE-like and the ADE-like SLs (ADE/CDE-like mut t1/2=255 min). RESULTS +11 20 mutations experimental_method Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +35 38 ADE structure_element Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +51 54 CDE structure_element Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +60 63 SLs structure_element Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +93 96 ADE structure_element Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +102 105 mut protein_state Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +106 110 t1/2 evidence Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +120 123 CDE structure_element Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +129 132 mut protein_state Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +133 137 t1/2 evidence Individual mutations of either the ADE-like or the CDE-like SLs showed intermediate effects (ADE-like mut t1/2=170 min, CDE-like mut t1/2=167 min), respectively. RESULTS +133 137 Ox40 protein These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells. RESULTS +138 142 mRNA chemical These findings underscore the importance of both structural motifs and reveal that they have an additive effect on the regulation of Ox40 mRNA expression in cells. RESULTS +7 40 structural and functional studies experimental_method Recent structural and functional studies have provided first insight into the RNA binding of Roquin. DISCUSS +78 81 RNA chemical Recent structural and functional studies have provided first insight into the RNA binding of Roquin. DISCUSS +93 99 Roquin protein Recent structural and functional studies have provided first insight into the RNA binding of Roquin. DISCUSS +0 10 Structures evidence Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +14 20 Roquin protein Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +21 29 bound to protein_state Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +30 33 CDE structure_element Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +34 36 SL structure_element Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +37 41 RNAs chemical Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +84 86 SL structure_element Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +87 90 RNA chemical Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +108 114 A-site site Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +122 139 N-terminal region structure_element Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +147 153 Roquin protein Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +221 257 pyrimidine–purine–pyrimidine triloop structure_element Structures of Roquin bound to CDE SL RNAs indicated mainly shape recognition of the SL RNA in the so-called A-site of the N-terminal region of the Roquin protein with no sequence specificity, except the requirement for a pyrimidine–purine–pyrimidine triloop. DISCUSS +21 24 CDE structure_element Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. DISCUSS +25 28 RNA chemical Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. DISCUSS +124 128 mRNA chemical Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. DISCUSS +152 158 Roquin protein Considering that the CDE RNA recognition is mostly structure specific and not sequence dependent, a wide spectrum of target mRNA might be recognized by Roquin. DISCUSS +18 30 SELEX assays experimental_method Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +51 72 RNA recognition motif structure_element Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +76 84 Roquin-1 protein Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +110 114 Ox40 protein Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +115 121 3′-UTR structure_element Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +166 173 3′-UTRs structure_element Here we have used SELEX assays to identify a novel RNA recognition motif of Roquin-1, which is present in the Ox40 3′-UTR and variations of which may be found in the 3′-UTRs of many other genes. DISCUSS +31 36 SELEX experimental_method Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. DISCUSS +45 48 ADE structure_element Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. DISCUSS +116 119 CDE structure_element Our experiments show that this SELEX-derived ADE shows functional activity comparable to the previously established CDE motif. DISCUSS +4 7 ADE structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +12 16 Ox40 protein The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +17 20 ADE structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +26 28 SL structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +29 33 RNAs chemical The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +40 42 SL structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +56 64 hexaloop structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +78 85 triloop structure_element The ADE and Ox40 ADE-like SL RNAs adopt SL folds with a hexaloop instead of a triloop. DISCUSS +43 67 RNA-helical stem regions structure_element Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. DISCUSS +75 78 ROQ structure_element Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. DISCUSS +107 114 triloop structure_element Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. DISCUSS +119 127 hexaloop structure_element Notably, the recognition of the respective RNA-helical stem regions by the ROQ domain is identical for the triloop and hexaloop motifs. DISCUSS +13 29 U-rich hexaloops structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +37 40 ADE structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +45 48 ADE structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +54 56 SL structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +57 61 RNAs chemical However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +111 116 helix structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +117 119 α4 structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +124 130 strand structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +131 133 β3 structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +141 144 ROQ structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +175 182 triloop structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +183 186 CDE structure_element However, the U-rich hexaloops in the ADE and ADE-like SL RNAs mediate a number of additional contacts with the helix α4 and strand β3 in the ROQ domain that are absent in the triloop CDE (Fig. 3b–f). DISCUSS +33 41 hexaloop structure_element Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. DISCUSS +57 63 Tyr250 residue_name_number Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. DISCUSS +132 136 loop structure_element Of particular importance for the hexaloop recognition is Tyr250, which acts as a stabilizing element for the integrity of a defined loop conformation. DISCUSS +3 9 stacks bond_interaction It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). DISCUSS +34 42 hexaloop structure_element It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). DISCUSS +55 58 CDE structure_element It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). DISCUSS +59 66 triloop structure_element It stacks with nucleotides in the hexaloop but not the CDE triloop (Fig. 3b,c). DISCUSS +23 29 Tyr250 residue_name_number The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). DISCUSS +34 37 ADE structure_element The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). DISCUSS +47 51 mRNA chemical The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). DISCUSS +66 74 Roquin-1 protein The functional role of Tyr250 for ADE-mediated mRNA regulation by Roquin-1 is thus explained by our experiments (Fig. 5b,c). DISCUSS +19 35 U-rich hexaloops structure_element The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +83 86 ROQ structure_element The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +92 95 U10 residue_name_number The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +97 100 U11 residue_name_number The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +105 108 U13 residue_name_number The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +116 120 Ox40 protein The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +121 124 ADE structure_element The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +130 132 SL structure_element The preference for U-rich hexaloops depends on nucleotide-specific interactions of ROQ with U10, U11 and U13 in the Ox40 ADE-like SL. DISCUSS +30 33 ROQ structure_element Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). DISCUSS +57 68 replacement experimental_method Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). DISCUSS +72 75 U11 residue_name_number Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). DISCUSS +80 83 U13 residue_name_number Consistent with this, loss of ROQ binding is observed on replacement of U11 and U13 by other bases (Table 2). DISCUSS +53 56 RNA chemical In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +90 96 Roquin protein In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +122 125 ADE structure_element In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +130 133 CDE structure_element In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +139 143 RNAs chemical In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +209 212 RNA chemical In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +213 217 stem structure_element In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +254 272 hexa- and triloops structure_element In spite of these differences in some aspects of the RNA recognition, overall features of Roquin targets are conserved in ADE and CDE-like RNAs, namely, a crucial role of non-sequence-specific contacts to the RNA stem and mainly shape recognition of the hexa- and triloops, respectively. DISCUSS +24 29 bound protein_state A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). DISCUSS +30 33 RNA chemical A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). DISCUSS +34 43 structure evidence A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). DISCUSS +60 78 tri- and hexaloops structure_element A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). DISCUSS +87 95 stacking bond_interaction A unique feature of the bound RNA structure, common to both tri- and hexaloops, is the stacking of a purine base onto the closing base pair (Fig. 3b,c). DISCUSS +9 24 structural data evidence Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. DISCUSS +79 85 Roquin protein Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. DISCUSS +111 113 SL structure_element Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. DISCUSS +114 117 RNA chemical Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. DISCUSS +150 155 loops structure_element Previous structural data and the results presented here therefore suggest that Roquin may recognize additional SL RNA motifs, potentially with larger loops. DISCUSS +19 24 SELEX experimental_method Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +53 66 U-rich motifs structure_element Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +141 153 U-rich loops structure_element Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +190 226 crosslinking and immunoprecipitation experimental_method Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +230 238 Roquin-1 protein Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +245 253 PAR-CLIP experimental_method Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +306 321 U-rich hexaloop structure_element Interestingly, the SELEX-derived motif resembles the U-rich motifs that were identified recently by Murakawa et al.. In their study, several U-rich loops of various sizes were identified by crosslinking and immunoprecipitation of Roquin-1 using PAR-CLIP and the data also included sequences comprising the U-rich hexaloop identified in our present work. DISCUSS +112 118 Roquin protein Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. DISCUSS +132 148 structural study experimental_method Most probably, the experimental setup of Murakawa et al. revealed both high- and low-affinity target motifs for Roquin, whereas our structural study reports on a high-affinity binding motif. DISCUSS +43 49 Roquin protein Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. DISCUSS +60 64 Ox40 protein Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. DISCUSS +73 76 Tnf protein Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. DISCUSS +77 84 3′-UTRs structure_element Notably, Murakawa et al. neither found the Roquin-regulated Ox40 nor the Tnf 3′-UTRs, as both genes are not expressed in HEK 293 cells. DISCUSS +46 48 SL structure_element However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +60 66 3′-UTR structure_element However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +70 73 A20 protein However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +74 78 mRNA chemical However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +108 114 Roquin protein However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +177 180 CDE structure_element However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +181 188 triloop structure_element However, their newly identified U-rich target SL within the 3′-UTR of A20 mRNA supports our conclusion that Roquin can accept alternative target motifs apart from the classical CDE triloop arrangement. DISCUSS +73 76 A20 protein It remains to be seen which exact features govern the recognition of the A20 SL by Roquin. DISCUSS +77 79 SL structure_element It remains to be seen which exact features govern the recognition of the A20 SL by Roquin. DISCUSS +83 89 Roquin protein It remains to be seen which exact features govern the recognition of the A20 SL by Roquin. DISCUSS +15 31 cis RNA elements structure_element The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. DISCUSS +35 42 3′-UTRs structure_element The regulatory cis RNA elements in 3′-UTRs may also be targeted by additional trans-acting factors. DISCUSS +32 44 endonuclease protein_type We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. DISCUSS +45 54 Regnase-1 protein We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. DISCUSS +72 78 Roquin protein We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. DISCUSS +129 134 mRNAs chemical We have recently identified the endonuclease Regnase-1 as a cofactor of Roquin function that shares an overlapping set of target mRNAs. DISCUSS +125 143 lipopolysaccharide chemical In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. DISCUSS +145 148 LPS chemical In another study, the overlap in targets was confirmed, but a mutually exclusive regulation was proposed based on studies in lipopolysaccharide (LPS)-stimulated myeloid cells. DISCUSS +16 22 Roquin protein In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +31 35 mRNA chemical In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +67 75 inactive protein_state In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +76 81 mRNAs chemical In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +89 98 Regnase-1 protein In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +107 111 mRNA chemical In these cells, Roquin induced mRNA decay only for translationally inactive mRNAs, while Regnase-1-induced mRNA decay depended on active translation of the target. DISCUSS +17 21 Ox40 protein In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. DISCUSS +76 84 Roquin-1 protein In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. DISCUSS +88 96 Roquin-2 protein In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. DISCUSS +151 161 deficiency experimental_method In CD4+ T cells, Ox40 does not show derepression in individual knockouts of Roquin-1 or Roquin-2 encoding genes, but is strongly induced upon combined deficiency of both genes. DISCUSS +25 36 deletion of experimental_method In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. DISCUSS +41 50 Regnase-1 protein In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. DISCUSS +73 77 Ox40 protein In addition, conditional deletion of the Regnase-1-encoding gene induced Ox40 expression in these cells. DISCUSS +25 29 Ox40 protein Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. DISCUSS +30 34 mRNA chemical Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. DISCUSS +38 44 Roquin protein Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. DISCUSS +48 55 Regnase protein_type Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. DISCUSS +243 270 double and triple knockouts experimental_method Whether induced decay of Ox40 mRNA by Roquin or Regnase proteins occurs in a mutually exclusive manner at different points during T-cell activation or shows cooperative regulation will have to await a direct comparison of T cells with single, double and triple knockouts of these genes. DISCUSS +38 42 Ox40 protein However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. DISCUSS +136 144 Roquin-1 protein However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. DISCUSS +149 157 Roquin-2 protein However, in cultures of CD4+ T cells, Ox40 is translated on day 4–5 and is expressed much higher in T cells with combined deficiency of Roquin-1 and Roquin-2. DISCUSS +45 59 reconstitution experimental_method At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +65 67 WT protein_state At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +68 76 Roquin-1 protein At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +102 106 Ox40 protein At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +169 175 active protein_state At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +176 180 mRNA chemical At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +184 192 Roquin-1 protein At this time point, the short-term inducible reconstitution with WT Roquin-1 was effective to reduced Ox40 expression, demonstrating the regulation of a translationally active mRNA by Roquin-1 in T cells (Fig. 5c). DISCUSS +44 52 Roquin-1 protein Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +56 64 Roquin-2 protein Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +98 102 Ox40 protein Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +103 107 mRNA chemical Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +111 116 EMSAs experimental_method Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +125 131 3′-UTR structure_element Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +135 139 Ox40 protein Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +197 218 filter binding assays experimental_method Recombinant N-terminal protein fragments of Roquin-1 or Roquin-2 bind with comparable affinity to Ox40 mRNA in EMSAs and the 3′-UTR of Ox40 is similarly retained by the two recombinant proteins in filter binding assays. DISCUSS +27 30 RNA chemical Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. DISCUSS +95 98 ADE structure_element Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. DISCUSS +103 106 CDE structure_element Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. DISCUSS +121 125 Ox40 protein Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. DISCUSS +126 132 3′-UTR structure_element Given the almost identical RNA contacts in both paralogues, we assume a similar recognition of ADE and CDE motifs in the Ox40 3′-UTR by both proteins. DISCUSS +39 48 Regnase-1 protein In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. DISCUSS +73 75 SL structure_element In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. DISCUSS +76 80 RNAs chemical In contrast, structural details on how Regnase-1 can interact with these SL RNAs are currently missing. DISCUSS +44 67 Regnase-1-binding sites site Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +71 119 crosslinking and immunoprecipitation experiments experimental_method Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +140 147 triloop structure_element Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +164 198 pyrimidine–purine–pyrimidine loops structure_element Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +218 223 stems structure_element Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +244 252 hexaloop structure_element Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +270 275 Ptgs2 gene Surprisingly, transcriptome-wide mapping of Regnase-1-binding sites in crosslinking and immunoprecipitation experiments identified specific triloop structures with pyrimidine–purine–pyrimidine loops in 3- to 7-nt-long stems, as well as a novel hexaloop structure in the Ptgs2 gene. DISCUSS +23 32 Regnase-1 protein Both were required for Regnase-1-mediated repression. DISCUSS +52 61 Regnase-1 protein These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner. DISCUSS +77 80 ADE structure_element These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner. DISCUSS +86 94 hexaloop structure_element These findings therefore raise the possibility that Regnase-1 interacts with ADE-like hexaloop structures either in a direct or indirect manner. DISCUSS +46 58 cis-elements structure_element Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. DISCUSS +93 96 SLs structure_element Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. DISCUSS +103 107 Ox40 protein Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. DISCUSS +111 115 Icos protein Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. DISCUSS +231 235 mRNA chemical Nevertheless, it becomes clear that composite cis-elements, that is, the presence of several SLs as in Ox40 or Icos, could attract multiple trans-acting factors that may potentially co-regulate or even act cooperatively to control mRNA expression through posttranscriptional pathways of gene regulation. DISCUSS +10 16 3′-UTR structure_element The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. DISCUSS +17 27 loop motif structure_element The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. DISCUSS +77 83 Roquin protein The novel 3′-UTR loop motif that we have identified as a bona fide target of Roquin now expands this multilayer mode of co-regulation. DISCUSS +43 47 mRNA chemical We suggest that differential regulation of mRNA expression is not only achieved through multiple regulators with individual preferences for a given motif or variants thereof, but that regulators may also identify and use distinct motifs, as long as they exhibit some basic features regarding shape, size and sequence. DISCUSS +35 42 3′-UTRs structure_element The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. DISCUSS +95 98 RNA chemical The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. DISCUSS +99 111 cis elements structure_element The presence of distinct motifs in 3′-UTRs offers a broader variability for gene regulation by RNA cis elements. DISCUSS +132 135 RNA chemical Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). DISCUSS +209 214 mRNAs chemical Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). DISCUSS +238 247 Staufen-1 protein Their accessibility can be modulated by trans-acting factors that may bind regulatory motifs, unfold higher-order structures in the RNA or maintain a preference for duplex structures as was shown recently for mRNAs that are recognized by Staufen-1 (ref.). DISCUSS +7 13 3′-UTR structure_element In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +21 25 Ox40 protein In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +26 30 mRNA chemical In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +44 47 ADE structure_element In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +61 64 CDE structure_element In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +70 72 SL structure_element In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +102 105 ROQ structure_element In the 3′-UTR of the Ox40 mRNA, we find one ADE-like and one CDE-like SL, with similar binding to the ROQ domain. DISCUSS +27 33 Roquin protein The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. DISCUSS +34 42 bound to protein_state The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. DISCUSS +47 51 Ox40 protein The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. DISCUSS +52 58 3′-UTR structure_element The exact stoichiometry of Roquin bound to the Ox40 3′-UTR is unknown. DISCUSS +24 46 secondary binding site site The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +51 56 dsRNA chemical The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +60 66 Roquin protein The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +68 74 B-site site The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +128 133 dsRNA chemical The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +168 174 Roquin protein The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +186 190 RNAs chemical The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +218 226 affinity evidence The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +227 230 SLs structure_element The recently identified secondary binding site for dsRNA in Roquin (B-site) could potentially allow for simultaneous binding of dsRNA and thereby promote engagement of Roquin and target RNAs before recognition of high-affinity SLs. DISCUSS +65 68 RNA chemical In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +87 90 Tnf protein In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +91 94 CDE structure_element In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +99 102 ADE structure_element In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +103 105 SL structure_element In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +106 110 RNAs chemical In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +139 154 crystal lattice evidence In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +208 213 dsRNA chemical In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +221 227 B site site In this respect, it is interesting to note that symmetry-related RNA molecules of both Tnf CDE and ADE SL RNAs are found in the respective crystal lattice in a position that corresponds to the recognition of dsRNA in the B site. DISCUSS +36 42 Roquin protein This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. DISCUSS +86 92 3′-UTR structure_element This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. DISCUSS +129 136 3′-UTRs structure_element This opens the possibility that one Roquin molecule may cluster two motifs in a given 3′-UTR and/or cluster motifs from distinct 3′-UTRs to enhance downstream processing. DISCUSS +19 21 SL structure_element Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +22 25 RNA chemical Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +70 76 Roquin protein Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +110 116 3′-UTR structure_element Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +124 130 Nfkbid protein Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +131 135 mRNA chemical Interestingly, two SL RNA elements that resemble bona fide ligands of Roquin have also been identified in the 3′-UTR of the Nfkbid mRNA. DISCUSS +58 71 binding sites site We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. DISCUSS +136 142 Roquin protein We therefore hypothesize that the combination of multiple binding sites may be more commonly used to enhance the functional activity of Roquin. DISCUSS +37 49 cis elements structure_element At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. DISCUSS +114 126 cis elements structure_element At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. DISCUSS +138 146 affinity evidence At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. DISCUSS +172 178 Roquin protein At the same time, the combination of cis elements may be important for differential gene regulation, as composite cis elements with lower affinity may be less sensitive to Roquin. DISCUSS +143 149 Roquin protein This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. DISCUSS +153 158 MALT1 protein This will lead to less effective repression in T cells when antigen recognition is of moderate signal strength and only incomplete cleavage of Roquin by MALT1 occurs. DISCUSS +46 52 3′-UTR structure_element For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms. DISCUSS +140 189 cross-linking and immunoprecipitation experiments experimental_method For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms. DISCUSS +202 219 RNA-binding sites site For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms. DISCUSS +225 243 structural biology experimental_method For understanding the intricate complexity of 3′-UTR regulation, future work will be necessary by combining large-scale approaches, such as cross-linking and immunoprecipitation experiments to identify RNA-binding sites, and structural biology to dissect the underlying molecular mechanisms. DISCUSS +0 5 SELEX experimental_method SELEX identifies a novel SL RNA ligand of Roquin-1. FIG +25 27 SL structure_element SELEX identifies a novel SL RNA ligand of Roquin-1. FIG +28 31 RNA chemical SELEX identifies a novel SL RNA ligand of Roquin-1. FIG +42 50 Roquin-1 protein SELEX identifies a novel SL RNA ligand of Roquin-1. FIG +41 49 Roquin-1 protein (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +71 76 2–440 residue_range (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +81 95 Roquin-1 M199R mutant (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +117 122 2–440 residue_range (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +164 167 ADE structure_element (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +190 194 Ox40 protein (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +195 201 3′-UTR structure_element (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +224 228 MEME experimental_method (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +244 249 SELEX experimental_method (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +259 262 RNA chemical (a) Enriched hexamers that were found by Roquin-1 N terminus (residues 2–440) or Roquin-1 M199R N terminus (residues 2–440) (see also Supplementary Fig. 1). (b) An ADE sequence motif in the Ox40 3′-UTR closely resembles the MEME motif found in SELEX-enriched RNA sequences. FIG +39 43 Ox40 protein (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. FIG +44 51 3′-UTRs structure_element (c) Conservation of the motif found in Ox40 3′-UTRs for various species as indicated. FIG +0 3 rn5 gene rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +41 44 rat taxonomy_domain rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +46 62 Rattus novegicus species rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +111 116 SELEX experimental_method rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +135 137 SL structure_element rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +139 142 ADE structure_element rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +151 155 Ox40 protein rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +156 159 ADE structure_element rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +165 173 hexaloop structure_element rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +174 176 SL structure_element rn5 is the fifth assembly version of the rat (Rattus novegicus). (d) Schematic representation of the predicted SELEX-derived consensus SL, ADE and the Ox40 ADE-like hexaloop SL. FIG +49 52 ADE structure_element The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. FIG +53 55 SL structure_element The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. FIG +77 101 non-Watson–Crick pairing bond_interaction The broken line between the G–G base pair in the ADE SL indicates a putative non-Watson–Crick pairing. FIG +4 8 Ox40 protein The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +9 12 CDE structure_element The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +18 20 SL structure_element The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +29 32 Tnf protein The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +33 36 CDE structure_element The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +37 39 SL structure_element The Ox40 CDE-like SL and the Tnf CDE SL are shown for comparison. FIG +0 3 NMR experimental_method NMR analysis of the SL RNAs used in this study. FIG +20 22 SL structure_element NMR analysis of the SL RNAs used in this study. FIG +23 27 RNAs chemical NMR analysis of the SL RNAs used in this study. FIG +40 46 1H NMR experimental_method Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +47 54 spectra evidence Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +66 69 ADE structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +70 72 SL structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +82 86 Ox40 protein Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +87 90 ADE structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +96 98 SL structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +111 115 Ox40 protein Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +116 119 CDE structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +125 127 SL structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +142 146 free protein_state Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +147 151 RNAs chemical Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +164 179 in complex with protein_state Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +184 192 Roquin-1 protein Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +193 196 ROQ structure_element Imino proton regions of one-dimensional 1H NMR spectra of (a) the ADE SL (b), the Ox40 ADE-like SL and (c) the Ox40 CDE-like SL are shown for free RNAs (black) and in complex with the Roquin-1 ROQ domain (red). FIG +15 17 SL structure_element The respective SL RNAs and their base pairs are indicated. FIG +18 22 RNAs chemical The respective SL RNAs and their base pairs are indicated. FIG +23 26 NMR experimental_method Red asterisks indicate NMR signals of the protein. FIG +82 85 NMR experimental_method Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. FIG +86 93 signals evidence Green lines in the secondary structure schemes on the left refer to visible imino NMR signals and thus experimental confirmation of the base pairs indicated. FIG +30 32 G6 residue_name_number The dotted green line between G6 and G15 in a highlights a G–G base pair. FIG +37 40 G15 residue_name_number The dotted green line between G6 and G15 in a highlights a G–G base pair. FIG +59 60 G residue_name The dotted green line between G6 and G15 in a highlights a G–G base pair. FIG +61 62 G residue_name The dotted green line between G6 and G15 in a highlights a G–G base pair. FIG +0 9 Structure evidence Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +17 25 Roquin-1 protein Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +26 29 ROQ structure_element Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +37 45 bound to protein_state Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +46 50 Ox40 protein Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +51 54 ADE structure_element Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +60 63 RNA chemical Structure of the Roquin-1 ROQ domain bound to Ox40 ADE-like RNA. FIG +32 49 crystal structure evidence (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +57 60 ROQ structure_element (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +78 85 174–325 residue_range (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +101 105 Ox40 protein (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +106 109 ADE structure_element (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +115 117 SL structure_element (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +118 121 RNA chemical (a) Cartoon presentation of the crystal structure of the ROQ domain (residues 174–325; blue) and the Ox40 ADE-like SL RNA (magenta). FIG +9 12 RNA chemical Selected RNA bases and protein secondary structure elements are labelled. FIG +25 29 Ox40 protein (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +30 33 ADE structure_element (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +39 41 SL structure_element (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +56 59 RNA chemical (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +60 68 hexaloop structure_element (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +123 132 structure evidence (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +140 151 ROQ-Tnf CDE complex_assembly (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +182 185 RNA chemical (b) Close-up view of the Ox40 ADE-like SL (bases in the RNA hexaloop are shown in magenta) and (c) the previously reported structure of the ROQ-Tnf CDE complex (bases of the triloop RNA are shown in green). FIG +5 29 RNA-interacting residues site Only RNA-interacting residues that are different in both structures are shown. FIG +57 67 structures evidence Only RNA-interacting residues that are different in both structures are shown. FIG +48 52 RNAs chemical Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +173 176 ROQ structure_element Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +200 203 U11 residue_name_number Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +208 211 U13 residue_name_number Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +219 223 Ox40 protein Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +224 227 ADE structure_element Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +233 235 SL structure_element Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +236 239 RNA chemical Both protein chains and remaining parts of both RNAs are shown in grey and protein residue side chains are shown in turquoise. (d) Close-up view of the contacts between the ROQ domain and nucleotides U11 and U13 of the Ox40 ADE-like SL RNA. FIG +52 57 helix structure_element The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +58 60 α4 structure_element The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +62 68 Tyr250 residue_name_number The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +73 79 Ser253 residue_name_number The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +108 114 strand structure_element The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +115 117 β3 structure_element The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +119 125 Phe255 residue_name_number The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +130 136 Val257 residue_name_number The nucleotides interact with the C-terminal end of helix α4 (Tyr250 and Ser253) and the N-terminal part of strand β3 (Phe255 and Val257). FIG +48 51 RNA chemical The protein chain is shown in turquoise and the RNA is shown in grey. FIG +46 49 ROQ structure_element (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +73 76 U10 residue_name_number (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +78 81 U11 residue_name_number (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +86 89 U13 residue_name_number (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +97 100 RNA chemical (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +101 109 hexaloop structure_element (e) Close-up view of the contacts between the ROQ domain and nucleotides U10, U11 and U13 in the RNA hexaloop. FIG +0 3 U11 residue_name_number U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +8 11 U13 residue_name_number U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +42 47 helix structure_element U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +48 50 α4 structure_element U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +61 67 Tyr250 residue_name_number U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +72 78 Gln247 residue_name_number U11 and U13 contact the C-terminal end of helix α4: residues Tyr250 and Gln247. FIG +18 24 Tyr250 residue_name_number The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. FIG +31 55 hydrophobic interactions bond_interaction The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. FIG +90 93 U10 residue_name_number The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. FIG +110 113 U11 residue_name_number The side chain of Tyr250 makes hydrophobic interactions with the pyrimidine side chain of U10 on one side and U11 on the other side. FIG +0 6 Lys259 residue_name_number Lys259 interacts with the phosphate groups of U10 and U11. FIG +46 49 U10 residue_name_number Lys259 interacts with the phosphate groups of U10 and U11. FIG +54 57 U11 residue_name_number Lys259 interacts with the phosphate groups of U10 and U11. FIG +25 48 hydrophobic interaction bond_interaction (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +57 63 Val257 residue_name_number (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +68 71 U11 residue_name_number (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +95 108 hydrogen bond bond_interaction (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +112 118 Lys259 residue_name_number (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +144 147 U10 residue_name_number (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +152 155 U11 residue_name_number (f) Close-up view of the hydrophobic interaction between Val257 and U11, as well as the double hydrogen bond of Lys259 with phosphate groups of U10 and U11. FIG +0 3 NMR experimental_method NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +16 19 ROQ structure_element NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +49 53 Ox40 protein NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +54 57 ADE structure_element NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +63 71 hexaloop structure_element NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +72 75 RNA chemical NMR analysis of ROQ domain interactions with the Ox40 ADE-like hexaloop RNA. FIG +4 11 Overlay experimental_method (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +15 26 1H,15N HSQC experimental_method (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +27 34 spectra evidence (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +49 53 free protein_state (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +54 57 ROQ structure_element (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +66 73 171–326 residue_range (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +85 100 in complex with protein_state (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +131 135 Ox40 protein (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +136 139 ADE structure_element (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +145 147 SL structure_element (a) Overlay of 1H,15N HSQC spectra of either the free ROQ domain (171–326, black) or in complex with stoichiometric amounts of the Ox40 ADE-like SL (red). FIG +12 33 chemical shift change evidence (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. FIG +63 66 ROQ structure_element (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. FIG +84 91 171–326 residue_range (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. FIG +163 170 spectra evidence (b) Plot of chemical shift change versus residue number in the ROQ domain (residues 171–326) from a. Grey negative bars indicate missing assignments in one of the spectra. FIG +14 22 prolines residue_name Gaps indicate prolines. FIG +4 11 Overlay experimental_method (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +19 22 ROQ structure_element (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +30 35 alone protein_state (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +47 62 in complex with protein_state (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +67 71 Ox40 protein (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +72 75 ADE structure_element (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +81 83 SL structure_element (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +97 101 Ox40 protein (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +102 105 CDE structure_element (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +111 113 SL structure_element (c) Overlay of the ROQ domain alone (black) or in complex with the Ox40 ADE-like SL (red) or the Ox40 CDE-like SL (green). FIG +0 19 Mutational analysis experimental_method Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +23 31 Roquin-1 protein Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +50 54 Ox40 protein Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +55 58 ADE structure_element Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +64 66 SL structure_element Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +71 75 Ox40 protein Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +76 82 3′-UTR structure_element Mutational analysis of Roquin-1-interactions with Ox40 ADE-like SL and Ox40 3′-UTR. FIG +4 14 EMSA assay experimental_method (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +40 49 wild-type protein_state (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +61 66 Y250A mutant (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +67 73 mutant protein_state (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +74 77 ROQ structure_element (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +104 108 Ox40 protein (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +109 112 ADE structure_element (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +118 120 SL structure_element (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +156 159 Tnf protein (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +160 163 CDE structure_element (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +164 166 SL structure_element (a) EMSA assay comparing binding of the wild-type and of the Y250A mutant ROQ domain for binding to the Ox40 ADE-like SL (left) or the previously described Tnf CDE SL (right). FIG +160 174 Flow cytometry experimental_method A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +178 182 Ox40 protein A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +187 191 Icos protein A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +234 239 Rc3h1 gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +240 243 2fl gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +244 246 fl gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +267 271 mice taxonomy_domain A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +285 294 tamoxifen chemical A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +312 317 Rc3h1 gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +318 321 2fl gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +322 324 fl gene A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +325 333 deletion experimental_method A comparison of further mutants is shown in Supplementary Fig. 4. (b) Schematic overview of the timeline used for the reconstitution experiment shown in c. (c) Flow cytometry of Ox40 and Icos surface expression on CD4+ Th1 cells from Rc3h1/2fl/fl; Cd4-Cre-ERT2; rtTA mice treated with tamoxifen (+tam) to induce Rc3h1/2fl/fl deletion or left untreated (− tam). FIG +74 84 retrovirus taxonomy_domain The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +98 109 doxycycline chemical The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +141 149 Roquin-1 protein The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +150 152 WT protein_state The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +154 162 Roquin-1 protein The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +163 168 Y250A mutant The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +172 180 Roquin-1 protein The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +181 186 K220A mutant The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +188 193 K239A mutant The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +198 203 R260A mutant The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +204 211 mutants protein_state The cells were then either left untransduced (UT) or were transduced with retrovirus containing a doxycycline-inducible cassette, to express Roquin-1 WT, Roquin-1 Y250A or Roquin-1 K220A, K239A and R260A mutants (see also Supplementary Fig. 5). FIG +25 33 Roquin-1 protein Functional importance of Roquin-1 target motifs in cells. FIG +20 24 Ox40 protein (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +25 31 3′-UTR structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +36 45 truncated protein_state (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +46 53 mutated protein_state (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +83 87 EMSA experimental_method (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +134 138 Ox40 protein (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +155 159 EMSA experimental_method (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +208 216 Roquin-1 protein (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +245 250 2–440 residue_range (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +276 285 wild-type protein_state (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +286 290 Ox40 protein (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +291 297 3′-UTR structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +315 324 mutations experimental_method (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +332 335 CDE structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +341 343 SL structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +349 352 ADE structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +358 360 SL structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +369 372 SLs structure_element (a) Overview of the Ox40 3′-UTR and truncated/mutated versions thereof as used for EMSA assays in b and the expression experiments of Ox40 in c and d. (b) EMSA experiments probing the interaction between the Roquin-1 N-terminal region (residues 2–440) and either the complete wild-type Ox40 3′-UTR or versions with mutations of the CDE-like SL, the ADE-like SL or both SLs (see a). FIG +148 156 Roquin-1 protein It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. FIG +166 172 3′-UTR structure_element It is noteworthy that the higher bands observed at large protein concentrations are probably additional nonspecific, lower-affinity interactions of Roquin-1 with the 3′-UTR or protein aggregates. FIG +13 17 Ox40 protein (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. FIG +18 53 MFI normalized to expression levels evidence (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. FIG +63 67 Ox40 protein (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. FIG +68 71 CDS structure_element (c) Relative Ox40 MFI normalized to expression levels from the Ox40 CDS construct. FIG +31 34 CDS structure_element Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +36 40 1–40 residue_range Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +42 46 1–80 residue_range Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +48 53 1–120 residue_range Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +58 69 full-length protein_state Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +77 80 ADE structure_element Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +86 89 mut protein_state Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +94 97 CDE structure_element Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +98 101 mut protein_state Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +113 123 double mut protein_state Error bars show s.d. of seven (CDS, 1–40, 1–80, 1–120 and full-length), six (ADE-like mut and CDE mut) or three (double mut) independent experiments. FIG +43 71 one-way analysis of variance experimental_method Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). FIG +73 78 ANOVA experimental_method Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). FIG +80 99 Kruskal–Wallis test experimental_method Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). FIG +112 143 Dunn’s multiple comparison test experimental_method Statistical significance was calculated by one-way analysis of variance (ANOVA) Kruskal–Wallis test followed by Dunn’s multiple comparison test (**P<0.01). FIG +4 21 mRNA decay curves evidence (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +67 79 retroviruses taxonomy_domain (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +91 95 Ox40 protein (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +96 99 CDS structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +108 114 3′-UTR structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +116 119 CDS structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +132 136 Ox40 protein (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +137 140 CDS structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +150 159 wild-type protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +160 166 3′-UTR structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +168 179 full length protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +194 198 Ox40 protein (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +199 210 full length protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +216 223 mutated protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +224 227 ADE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +240 243 ADE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +249 252 mut protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +266 270 Ox40 protein (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +271 282 full length protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +288 295 mutated protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +296 299 CDE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +312 315 CDE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +321 324 mut protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +341 345 Ox40 protein (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +346 357 full length protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +363 370 mutated protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +371 374 ADE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +379 382 CDE structure_element (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +391 401 Double mut protein_state (d) mRNA decay curves of Hela Tet-Off cells stably transduced with retroviruses expressing Ox40 CDS without 3′-UTR (CDS, red line), Ox40 CDS with its wild-type 3′-UTR (full length, black line), Ox40 full length with mutated ADE-like motif (ADE-like mut, grey line), Ox40 full length with mutated CDE-like motif (CDE-like mut, green line) or Ox40 full length with mutated ADE and CDE motifs (Double mut, blue line). FIG +0 20 mRNA half-life times evidence mRNA half-life times were calculated with Graph Pad Prism. FIG +0 41 Data collection and refinement statistics evidence Data collection and refinement statistics. TABLE +2 5 ROQ structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +6 10 Ox40 protein "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +10 13 ADE structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +19 21 SL structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +22 25 ROQ structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +26 29 ADE structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +30 32 SL structure_element "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +642 670 Root mean squared deviations evidence "  ROQ-Ox40ADE-like SL ROQ-ADE SL Data collection  space group P21212 P212121        Cell dimensions  a, b, c (Å) 89.66, 115.79, 42.61 72.90, 89.30, 144.70  α, β, γ (°) 90, 90, 90 90, 90, 90  Resolution (Å) 50–2.23 (2.29–2.23) 50–3.0 (3.08–3.00)  Rmerge 5.9 (68.3) 14.8 (93.8)  I/σI 14.9 (2.1) 16.7 (3.1)  Completeness (%) 98.7 (97.7) 99.9 (99.9)  Redundancy 3.9 (3.7) 13.2 (12.7)       Refinement  Resolution (Å) 2.23 3.00  No. reflections 21,018 18,598  Rwork/Rfree 21.8/25.7 18.6/23.4        No. atoms  Protein 2,404 4,820  Ligand/ion 894 1,708  Water 99 49  B-factor overall 47.2 60.4       Root mean squared deviations  Bond lengths (Å) 0.006 0.014  Bond angles (°) 1.07 1.77       Ramachandran plot  Most favoured (%) 98.6 99.8  Additional allowed (%) 1.4 0.2 " TABLE +0 3 ADE structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +5 30 alternative decay element structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +32 35 CDE structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +37 63 constitutive decay element structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +65 67 SL structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +69 78 stem loop structure_element ADE, alternative decay element; CDE, constitutive decay element; SL, stem loop. TABLE +28 35 crystal evidence For each data set, only one crystal has been used. TABLE +0 2 KD evidence KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1. TABLE +16 20 RNAs chemical KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1. TABLE +35 51 SPR measurements experimental_method KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1. TABLE +69 72 ROQ structure_element KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1. TABLE +83 91 Roquin-1 protein KD for selected RNAs obtained from SPR measurements with immobilized ROQ domain of Roquin-1. TABLE