cometadata/datacite-titles-descriptions-related-identifiers huggingface dataset:
DOI: 10.6084/m9.figshare.16651916.v2
title: <i>In vivo</i> discovery of RNA proximal proteins via proximity-dependent biotinylation

description: RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins <i>in vivo</i> via proximity-based biotinylation. RPL applied to <i>U1</i> identified proteins involved in both <i>U1</i> canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A)<sup>+</sup> RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA–protein interactions important for health and disease.
DOI: 10.6084/m9.figshare.14507167.v1
title: RGG-motif containing mRNA export factor Gbp2 acts as a translation repressor

description: Complex cascades of RNA-binding proteins regulate the mRNA metabolism and influence gene expression. Several distinct proteins act at different stages of mRNA life cycle. SR family proteins in yeast are implicated in mRNA processing and nuclear export. In this report, we uncover the role of an SR/RGG-motif containing mRNA export factor Gbp2 in mRNA translation regulation. We demonstrate that Gbp2 localizes to cytoplasmic granules upon heat shock and oxidative stress. Our pull-down assays demonstrate that Gbp2 directly binds to the conserved translation factor eIF4G1 via its RGG motif. We further mapped the region on eIF4G1 to which Gbp2 binds and observed that the binding region overlaps with another translation repressor Sbp1. We found that the RGG-motif deletion mutant is defective in localizing to polysome fractions. Upon tethering Gbp2 to a GFP reporter mRNA <i>in vivo</i>, translation of GFP reporter decreased significantly indicating that Gbp2 acts as a translation repressor. Consistent with these results, we show that Gbp2 can directly repress mRNA translation in the <i>in vitro</i> translation systems in an RGG-motif dependent manner. Taken together, our results establish that the mRNA export factor Gbp2 has a vital role in repressing translation of mRNA. We propose that Gbp2 is a multifaceted RGG-motif protein responsible for translational repression without affecting mRNA levels.
DOI: 10.6084/m9.figshare.14528858.v1
title: Optimization of a fluorescent-mRNA based real-time assay for precise kinetic measurements of ribosomal translocation

description: Kinetic characterization of ribosomal translocation is important for understanding the mechanism of elongation in protein synthesis. Here we have optimized a popular fluorescent-mRNA based translocation assay conducted in stopped-flow, by calibrating it with the functional tripeptide formation assay in quench-flow. We found that a fluorescently labelled mRNA, ten bases long from position +1 (mRNA+10), is best suited for both assays as it forms tripeptide at a fast rate equivalent to the longer mRNAs, and yet produces a large fluorescence change upon mRNA movement. Next, we compared the commonly used peptidyl tRNA analog, N-acetyl-Phe-tRNA<sup>Phe</sup>, with the natural dipeptidyl fMet-Phe-tRNA<sup>Phe</sup> in the stopped-flow assay. This analog translocates about two times slower than the natural dipeptidyl tRNA and produces biphasic kinetics. The rates reduce further at lower temperatures and with higher Mg<sup>2+</sup> concentration, but improve with higher elongation factor G (EF-G) concentration, which increase both rate and amplitude of the fast phase significantly. In summary, we present here an improved real time assay for monitoring mRNA-translocation with the natural- and an N-Ac-analog of dipeptidyl tRNA.
DOI: 10.6084/m9.figshare.14184593.v2
title: Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes

description: Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in <i>Escherichia coli</i>. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique <i>dus</i> gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using <i>in vitro</i> activity tests as well as <i>in vivo E. coli</i> complementation assays with the enzyme from <i>Mycoplasma capricolum</i> (DusB<i><sub>MCap</sub></i>), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusB<i><sub>MCap</sub></i> modifies U17 to D17 but also synthetizes D20/D20a combining therefore both <i>E. coli</i> DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the <i>M. capricolum</i> DusB enzyme could be an ancestral property.
DOI: 10.6084/m9.figshare.14536001
title: The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners

description: The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5ʹ end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in <i>Leishmania</i> species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in <i>L. infantum</i>. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in <i>Trypanosoma brucei</i>, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the <i>Leishmania</i>-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in <i>Leishmania</i> (W45) did not substantially impair the identified interactions. Finally, the crystal structure of <i>Leishmania</i> EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its <i>Trypanosoma</i> orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.
DOI: 10.6084/m9.figshare.14184593
title: Dihydrouridine synthesis in tRNAs is under reductive evolution in Mollicutes

description: Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in <i>Escherichia coli</i>. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique <i>dus</i> gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using <i>in vitro</i> activity tests as well as <i>in vivo E. coli</i> complementation assays with the enzyme from <i>Mycoplasma capricolum</i> (DusB<i><sub>MCap</sub></i>), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusB<i><sub>MCap</sub></i> modifies U17 to D17 but also synthetizes D20/D20a combining therefore both <i>E. coli</i> DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the <i>M. capricolum</i> DusB enzyme could be an ancestral property.
DOI: 10.6084/m9.figshare.14197333
title: Identification of conserved slow codons that are important for protein expression and function

description: Due to the redundancy of the genetic code most amino acids are encoded by several ‘synonymous‘ codons. These codons are used unevenly, and each organism demonstrates its own unique codon usage bias, where the ‘preferred’ codons are associated with tRNAs that are found in high concentrations. Therefore, for decades, the prevailing view had been that preferred and non-preferred codons are linked to high or slow translation rates, respectively. However, this simplified view is contrasted by the frequent failures of codon-optimization efforts and by evidence of non-preferred (<i>i.e</i>. ‘slow’) codons having specific roles important for efficient production of functional proteins. One such specific role of slower codons is the regulation of co-translational protein folding, a complex biophysical process that is very challenging to model or to measure. Here, we combined a genome-wide approach with experiments to investigate the role of slow codons in protein production and co-translational folding. We analysed homologous gene groups from divergent bacteria and identified positions of inter-species conservation of bias towards slow codons. We then generated mutants where the conserved slow codons are substituted with ‘fast’ ones, and experimentally studied the effects of these codon substitutions. Using cellular and biochemical approaches we find that at certain locations, slow-to-fast codon substitutions reduce protein expression, increase protein aggregation, and impair protein function. This report provides an approach for identifying functionally relevant regions with slower codons and demonstrates that such codons are important for protein expression and function.
DOI: 10.6084/m9.figshare.14536001.v1
title: The translation initiation factor EIF4E5 from Leishmania: crystal structure and interacting partners

description: The mRNA cap-binding protein, eIF4E, mediates the recognition of the mRNA 5ʹ end and, as part of the heterotrimeric eIF4F complex, facilitates the recruitment of the ribosomal subunits to initiate eukaryotic translation. Various regulatory events involving eIF4E and a second eIF4F subunit, eIF4G, are required for proper control of translation initiation. In pathogenic trypanosomatids, six eIF4Es and five eIF4Gs have been described, several forming different eIF4F-like complexes with yet unresolved roles. EIF4E5 is one of the least known of the trypanosomatid eIF4Es and has not been characterized in <i>Leishmania</i> species. Here, we used immunoprecipitation assays, combined with mass-spectrometry, to identify major EIF4E5 interacting proteins in <i>L. infantum</i>. A constitutively expressed, HA-tagged, EIF4E5 co-precipitated mainly with EIF4G1 and binding partners previously described in <i>Trypanosoma brucei</i>, EIF4G1-IP, RBP43 and the 14-3-3 proteins. In contrast, no clear co-precipitation with EIF4G2, also previously reported, was observed. EIF4E5 also co-precipitated with protein kinases, possibly associated with cell-cycle regulation, selected RNA binding proteins and histones. Phosphorylated residues were identified and mapped to the <i>Leishmania</i>-specific C-terminal end. Mutagenesis of the tryptophan residue (W53) postulated to mediate interactions with protein partners or of a neighbouring tryptophan conserved in <i>Leishmania</i> (W45) did not substantially impair the identified interactions. Finally, the crystal structure of <i>Leishmania</i> EIF4E5 evidences remarkable differences in the eIF4G interfacing region, when compared with human eIF4E-1 and with its <i>Trypanosoma</i> orthologue. Mapping of its C-terminal end near the cap-binding site also imply relevant differences in cap-binding function and/or regulation.
DOI: 10.6084/m9.figshare.14776249
title: A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics

description: Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in <i>Saccharomyces cerevisiae</i> that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.
DOI: 10.6084/m9.figshare.16651916.v1
title: <i>In vivo</i> discovery of RNA proximal proteins via proximity-dependent biotinylation

description: RNA molecules function as messenger RNAs (mRNAs) that encode proteins and noncoding transcripts that serve as adaptor molecules, structural components, and regulators of genome organization and gene expression. Their function and regulation are largely mediated by RNA binding proteins (RBPs). Here we present RNA proximity labelling (RPL), an RNA-centric method comprising the endonuclease-deficient Type VI CRISPR-Cas protein dCas13b fused to engineered ascorbate peroxidase APEX2. RPL discovers target RNA proximal proteins <i>in vivo</i> via proximity-based biotinylation. RPL applied to <i>U1</i> identified proteins involved in both <i>U1</i> canonical and noncanonical functions. Profiling of poly(A) tail proximal proteins uncovered expected categories of RBPs and provided additional evidence for 5ʹ-3ʹ proximity and unexplored subcellular localizations of poly(A)<sup>+</sup> RNA. Our results suggest that RPL allows rapid identification of target RNA binding proteins in native cellular contexts, and is expected to pave the way for discovery of novel RNA–protein interactions important for health and disease.
DOI: 10.6084/m9.figshare.14184593.v1
title: Dihydrouridine synthesis in tRNAs is under reductive evolution in mollicutes

description: Dihydrouridine (D) is a tRNA-modified base conserved throughout all kingdoms of life and assuming an important structural role. The conserved dihydrouridine synthases (Dus) carries out D-synthesis. DusA, DusB and DusC are bacterial members, and their substrate specificity has been determined in <i>Escherichia coli</i>. DusA synthesizes D20/D20a while DusB and DusC are responsible for the synthesis of D17 and D16, respectively. Here, we characterize the function of the unique <i>dus</i> gene encoding a DusB detected in Mollicutes, which are bacteria that evolved from a common Firmicute ancestor via massive genome reduction. Using <i>in vitro</i> activity tests as well as <i>in vivo E. coli</i> complementation assays with the enzyme from <i>Mycoplasma capricolum</i> (DusB<i><sub>MCap</sub></i>), a model organism for the study of these parasitic bacteria, we show that, as expected for a DusB homolog, DusB<i><sub>MCap</sub></i> modifies U17 to D17 but also synthetizes D20/D20a combining therefore both <i>E. coli</i> DusA and DusB activities. Hence, this is the first case of a Dus enzyme able to modify up to three different sites as well as the first example of a tRNA-modifying enzyme that can modify bases present on the two opposite sides of an RNA-loop structure. Comparative analysis of the distribution of DusB homologs in Firmicutes revealed the existence of three DusB subgroups namely DusB1, DusB2 and DusB3. The first two subgroups were likely present in the Firmicute ancestor, and Mollicutes have retained DusB1 and lost DusB2. Altogether, our results suggest that the multisite specificity of the <i>M. capricolum</i> DusB enzyme could be an ancestral property.
DOI: 10.6084/m9.figshare.14776249.v1
title: A small ribosome-associated ncRNA globally inhibits translation by restricting ribosome dynamics

description: Ribosome-associated non-coding RNAs (rancRNAs) have been recognized as an emerging class of regulatory molecules capable of fine-tuning translation in all domains of life. RancRNAs are ideally suited for allowing a swift response to changing environments and are therefore considered pivotal during the first wave of stress adaptation. Previously, we identified an mRNA-derived 18 nucleotides long rancRNA (rancRNA_18) in <i>Saccharomyces cerevisiae</i> that rapidly downregulates protein synthesis during hyperosmotic stress. However, the molecular mechanism of action remained enigmatic. Here, we combine biochemical, genetic, transcriptome-wide and structural evidence, thus revealing rancRNA_18 as global translation inhibitor by targeting the E-site region of the large ribosomal subunit. Ribosomes carrying rancRNA_18 possess decreased affinity for A-site tRNA and impaired structural dynamics. Cumulatively, these discoveries reveal the mode of action of a rancRNA involved in modulating protein biosynthesis at a thus far unequalled precision.
DOI: 10.6084/m9.figshare.14616895
title: Structural model of the M7G46 Methyltransferase TrmB in complex with tRNA

description: TrmB belongs to the class I S-adenosylmethionine (SAM)-dependent methyltransferases (MTases) and introduces a methyl group to guanine at position 7 (m<sup>7</sup>G) in tRNA. In tRNAs m<sup>7</sup>G is most frequently found at position 46 in the variable loop and forms a tertiary base pair with C13 and U22, introducing a positive charge at G46. The TrmB/Trm8 enzyme family is structurally diverse, as TrmB proteins exist in a monomeric, homodimeric, and heterodimeric form. So far, the exact enzymatic mechanism, as well as the tRNA-TrmB crystal structure is not known. Here we present the first crystal structures of <i>B. subtilis</i> TrmB in complex with SAM and SAH. The crystal structures of TrmB apo and in complex with SAM and SAH have been determined by X-ray crystallography to 1.9 Å (apo), 2.5 Å (SAM), and 3.1 Å (SAH). The obtained crystal structures revealed Tyr193 to be important during SAM binding and MTase activity. Applying fluorescence polarization, the dissociation constant K<sub>d</sub> of TrmB and tRNA<sup>Phe</sup> was determined to be 0.12 µM ± 0.002 µM. Luminescence-based methyltransferase activity assays revealed cooperative effects during TrmB catalysis with half-of-the-site reactivity at physiological SAM concentrations. Structural data retrieved from small-angle x-ray scattering (SAXS), mass-spectrometry of cross-linked complexes, and molecular docking experiments led to the determination of the TrmB-tRNA<sup>Phe</sup> complex structure.
DOI: 10.6084/m9.figshare.14883639
title: An integrated DNA and RNA variant detector identifies a highly conserved three base exon in the <i>MAP4K5</i> kinase locus

description: RNA variants that emerge from editing and alternative splicing form important regulatory stages in protein signalling. In this report, we apply an integrated DNA and RNA variant detection workbench to define the range of RNA variants that deviate from the reference genome in a human melanoma cell model. The RNA variants can be grouped into (i) classic ADAR-like or APOBEC-like RNA editing events and (ii) multiple-nucleotide variants (MNVs) including three and six base pair in-frame non-canonical unmapped exons. We focus on validating representative genes of these classes. First, clustered non-synonymous RNA edits (A-I) in the <i>CDK13</i> gene were validated by Sanger sequencing to confirm the integrity of the RNA variant detection workbench. Second, a highly conserved RNA variant in the <i>MAP4K5</i> gene was detected that results most likely from the splicing of a non-canonical three-base exon. The two RNA variants produced from the <i>MAP4K5</i> locus deviate from the genomic reference sequence and produce V569E or V569del isoform variants. Low doses of splicing inhibitors demonstrated that the MAP4K5-V569E variant emerges from an SF3B1-dependent splicing event. Mass spectrometry of the recombinant SBP-tagged MAP4K5<sup>V569E</sup> and MAP4K5<sup>V569del</sup> proteins pull-downs in transfected cell systems was used to identify the protein-protein interactions of these two MAP4K5 isoforms and propose possible functions. Together these data highlight the utility of this integrated DNA and RNA variant detection platform to detect RNA variants in cancer cells and support future analysis of RNA variant detection in cancer tissue.