PMC 20140719 pmc.key 4786784 CC BY no 0 0 10.1038/ncomms10950 ncomms10950 4786784 26952537 10950 This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/ surname:Agrawal;given-names:Anant A. surname:Salsi;given-names:Enea surname:Chatrikhi;given-names:Rakesh surname:Henderson;given-names:Steven surname:Jenkins;given-names:Jermaine L. surname:Green;given-names:Michael R. surname:Ermolenko;given-names:Dmitri N. surname:Kielkopf;given-names:Clara L. TITLE front 7 2016 0 An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:25Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:09:24Z U2AF65–RNA-binding domain site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site ABSTRACT abstract 79 How the essential pre-mRNA splicing factor U2AF65 recognizes the polypyrimidine (Py) signals of the major class of 3′ splice sites in human gene transcripts remains incompletely understood. We determined four structures of an extended U2AF65–RNA-binding domain bound to Py-tract oligonucleotides at resolutions between 2.0 and 1.5 Å. These structures together with RNA binding and splicing assays reveal unforeseen roles for U2AF65 inter-domain residues in recognizing a contiguous, nine-nucleotide Py tract. The U2AF65 linker residues between the dual RNA recognition motifs (RRMs) recognize the central nucleotide, whereas the N- and C-terminal RRM extensions recognize the 3′ terminus and third nucleotide. Single-molecule FRET experiments suggest that conformational selection and induced fit of the U2AF65 RRMs are complementary mechanisms for Py-tract association. Altogether, these results advance the mechanistic understanding of molecular recognition for a major class of splice site signals. protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T16:12:17Z pre-mRNA splicing factor protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:12:38Z polypyrimidine chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:12:46Z Py site SO: melaniev@ebi.ac.uk 2023-03-20T16:12:54Z 3′ splice sites species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:31Z determined four structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:25Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:09:24Z U2AF65–RNA-binding domain protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:43:11Z Py-tract oligonucleotides evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:47Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:13:51Z RNA binding and splicing assays protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-20T16:13:56Z inter-domain residues structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:02Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:49Z nucleotide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:21Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:29Z RNA recognition motifs structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions site SO: melaniev@ebi.ac.uk 2023-03-20T16:17:09Z 3′ terminus residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:33Z third chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:17:19Z Single-molecule FRET protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:03Z splice site ABSTRACT abstract 1090 The pre-mRNA splicing factor U2AF65 recognizes 3′ splice sites in human gene transcripts, but the details are not fully understood. Here, the authors report U2AF65 structures and single molecule FRET that reveal mechanistic insights into splice site recognition. protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T16:12:17Z pre-mRNA splicing factor protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-20T16:12:54Z 3′ splice sites species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:18:19Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:27Z single molecule FRET site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:03Z splice site INTRO paragraph 1356 The differential skipping or inclusion of alternatively spliced pre-mRNA regions is a major source of diversity for nearly all human gene transcripts. The splice sites are marked by relatively short consensus sequences and are regulated by additional pre-mRNA motifs (reviewed in ref.). At the 3′ splice site of the major intron class, these include a polypyrimidine (Py) tract comprising primarily Us or Cs, which is preceded by a branch point sequence (BPS) that ultimately serves as the nucleophile in the splicing reaction and an AG-dinucleotide at the 3′ splice site junction. Disease-causing mutations often compromise pre-mRNA splicing (reviewed in refs), yet a priori predictions of splice sites and the consequences of their mutations are challenged by the brevity and degeneracy of known splice site sequences. High-resolution structures of intact splicing factor–RNA complexes would offer key insights regarding the juxtaposition of the distinct splice site consensus sequences and their relationship to disease-causing point mutations. structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:49:28Z pre-mRNA regions species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human site SO: melaniev@ebi.ac.uk 2023-03-20T16:19:52Z splice sites structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:20:09Z short consensus sequences structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:20:13Z pre-mRNA motifs site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:20:18Z polypyrimidine (Py) tract residue_name SO: melaniev@ebi.ac.uk 2023-03-20T16:20:24Z U residue_name SO: melaniev@ebi.ac.uk 2023-03-20T16:20:27Z C site SO: melaniev@ebi.ac.uk 2023-03-20T16:20:05Z branch point sequence site SO: melaniev@ebi.ac.uk 2023-03-20T16:24:05Z BPS chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:20:35Z AG-dinucleotide site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:50:01Z pre-mRNA site SO: melaniev@ebi.ac.uk 2023-03-20T16:19:52Z splice sites site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:03Z splice site evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:20:45Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:30:59Z intact complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:20:56Z splicing factor–RNA site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:03Z splice site INTRO paragraph 2410 The early-stage pre-mRNA splicing factor U2AF65 is essential for viability in vertebrates and other model organisms (for example, ref.). A tightly controlled assembly among U2AF65, the pre-mRNA, and partner proteins sequentially identifies the 3′ splice site and promotes association of the spliceosome, which ultimately accomplishes the task of splicing. Initially U2AF65 recognizes the Py-tract splice site signal. In turn, the ternary complex of U2AF65 with SF1 and U2AF35 identifies the surrounding BPS and 3′ splice site junctions. Subsequently U2AF65 recruits the U2 small nuclear ribonucleoprotein particle (snRNP) and ultimately dissociates from the active spliceosome. protein_type MESH: melaniev@ebi.ac.uk 2023-03-20T16:12:17Z pre-mRNA splicing factor protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 taxonomy_domain DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:24:20Z vertebrates complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:24:24Z assembly protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:24:29Z pre-mRNA site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:26:09Z spliceosome protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:06Z Py-tract site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:03Z splice site complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:26:18Z ternary complex protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:25:48Z SF1 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:25:51Z U2AF35 site SO: melaniev@ebi.ac.uk 2023-03-20T16:24:06Z BPS site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:25:43Z U2 small nuclear ribonucleoprotein particle complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:25:55Z snRNP protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:26:02Z active complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:26:09Z spliceosome INTRO paragraph 3092 Biochemical characterizations of U2AF65 demonstrated that tandem RNA recognition motifs (RRM1 and RRM2) recognize the Py tract (Fig. 1a). Milestone crystal structures of the core U2AF65 RRM1 and RRM2 connected by a shortened inter-RRM linker (dU2AF651,2) detailed a subset of nucleotide interactions with the individual U2AF65 RRMs. A subsequent NMR structure characterized the side-by-side arrangement of the minimal U2AF65 RRM1 and RRM2 connected by a linker of natural length (U2AF651,2), yet depended on the dU2AF651,2 crystal structures for RNA interactions and an ab initio model for the inter-RRM linker conformation. As such, the molecular mechanisms for Py-tract recognition by the intact U2AF65–RNA-binding domain remained unknown. Here, we use X-ray crystallography and biochemical studies to reveal new roles in Py-tract recognition for the inter-RRM linker and key residues surrounding the core U2AF65 RRMs. We use single-molecule Förster resonance energy transfer (smFRET) to characterize the conformational dynamics of this extended U2AF65–RNA-binding domain during Py-tract recognition. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:29:00Z Biochemical characterizations protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:29Z RNA recognition motifs structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:57Z shortened structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:13Z dU2AF651,2 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs experimental_method MESH: melaniev@ebi.ac.uk 2023-06-15T14:16:43Z NMR evidence DUMMY: melaniev@ebi.ac.uk 2023-06-15T14:16:52Z structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:48:49Z linker protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:19Z natural length mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:13Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures chemical CHEBI: melaniev@ebi.ac.uk 2023-06-15T11:52:42Z RNA structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:30:59Z intact structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:09:24Z U2AF65–RNA-binding domain experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:10Z X-ray crystallography experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:13Z biochemical studies chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:17Z single-molecule Förster resonance energy transfer experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:31:30Z conformational dynamics protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:25Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:09:24Z U2AF65–RNA-binding domain chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract RESULTS title_1 4200 Results RESULTS title_2 4208 Cognate U2AF65–Py-tract recognition requires RRM extensions protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions RESULTS paragraph 4270 The RNA affinity of the minimal U2AF651,2 domain comprising the core RRM1–RRM2 folds (U2AF651,2, residues 148–336) is relatively weak compared with full-length U2AF65 (Fig. 1a,b; Supplementary Fig. 1). Historically, this difference was attributed to the U2AF65 arginine–serine rich domain, which contacts pre-mRNA–U2 snRNA duplexes outside of the Py tract. We noticed that the RNA-binding affinity of the U2AF651,2 domain was greatly enhanced by the addition of seven and six residues at the respective N and C termini of the minimal RRM1 and RRM2 (U2AF651,2L, residues 141–342; Fig. 1a). In a fluorescence anisotropy assay for binding a representative Py tract derived from the well-characterized splice site of the adenovirus major late promoter (AdML), the RNA affinity of U2AF651,2L increased by 100-fold relative to U2AF651,2 to comparable levels as full-length U2AF65 (Fig. 1b; Supplementary Fig. 1a–d). Likewise, both U2AF651,2L and full-length U2AF65 showed similar sequence specificity for U-rich stretches in the 5′-region of the Py tract and promiscuity for C-rich regions in the 3′-region (Fig. 1c, Supplementary Fig. 1e–h). evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:48:54Z folds mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:22Z 148–336 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:34:43Z arginine–serine rich domain complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:34:50Z pre-mRNA–U2 snRNA duplexes chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:03Z RNA-binding affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:14Z addition of seven and six residues protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:47Z 141–342 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:50Z fluorescence anisotropy assay chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:04Z splice site gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:35:56Z adenovirus major late promoter gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:15Z AdML evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:26Z U2AF65 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:36:28Z sequence specificity structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:36:32Z U-rich stretches site SO: melaniev@ebi.ac.uk 2023-03-20T16:36:40Z 5′-region chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:36:45Z C-rich regions site SO: melaniev@ebi.ac.uk 2023-03-20T16:36:52Z 3′-region RESULTS title_2 5425 U2AF65-bound Py tract comprises nine contiguous nucleotides protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:37:53Z U2AF65-bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:00Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:50:55Z nucleotides RESULTS paragraph 5485 To investigate the structural basis for cognate U2AF65 recognition of a contiguous Py tract, we determined four crystal structures of U2AF651,2L bound to Py-tract oligonucleotides (Fig. 2a; Table 1). By sequential boot strapping (Methods), we optimized the oligonucleotide length, the position of a Br-dU, and the identity of the terminal nucleotide (rU, dU and rC) to achieve full views of U2AF651,2L bound to contiguous Py tracts at up to 1.5 Å resolution. The protein and oligonucleotide conformations are nearly identical among the four new U2AF651,2L structures (Supplementary Fig. 2a). The U2AF651,2L RRM1 and RRM2 associate with the Py tract in a parallel, side-by-side arrangement (shown for representative structure iv in Fig. 2b,c; Supplementary Movie 1). An extended conformation of the U2AF65 inter-RRM linker traverses across the α-helical surface of RRM1 and the central β-strands of RRM2 and is well defined in the electron density (Fig. 2b). The extensions at the N terminus of RRM1 and C terminus of RRM2 adopt well-ordered α-helices. Both RRM1/RRM2 extensions and the inter-RRM linker of U2AF651,2L directly recognize the bound oligonucleotide. We compare the global conformation of the U2AF651,2L structures with the prior dU2AF651,2 crystal structure and U2AF651,2 NMR structure in the Supplementary Discussion and Supplementary Fig. 2. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:03Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:42:55Z determined evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:43:11Z Py-tract oligonucleotides experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:43:16Z sequential boot strapping chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:43:21Z oligonucleotide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:43:27Z Br-dU chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide residue_name SO: melaniev@ebi.ac.uk 2023-03-20T16:44:02Z rU residue_name SO: melaniev@ebi.ac.uk 2023-03-20T16:44:11Z dU residue_name SO: melaniev@ebi.ac.uk 2023-03-20T16:44:18Z rC mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:03Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:00Z Py tracts chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:44:37Z oligonucleotide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:44:41Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:01Z parallel protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:14Z extended conformation protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:45:21Z α-helical surface structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:45:33Z β-strands structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:40Z electron density structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:45:45Z extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:14Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:45:51Z α-helices structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:45:59Z extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:10Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:46:07Z oligonucleotide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:20Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:13Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:29Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 experimental_method MESH: melaniev@ebi.ac.uk 2023-06-15T14:17:19Z NMR evidence DUMMY: melaniev@ebi.ac.uk 2023-06-15T14:17:27Z structure RESULTS paragraph 6853 The discovery of nine U2AF65-binding sites for contiguous Py-tract nucleotides was unexpected. Based on dU2AF651,2 structures, we originally hypothesized that the U2AF65 RRMs would bind the minimal seven nucleotides observed in these structures. Surprisingly, the RRM2 extension/inter-RRM linker contribute new central nucleotide-binding sites near the RRM1/RRM2 junction and the RRM1 extension recognizes the 3′-terminal nucleotide (Fig. 2c; Supplementary Movie 1). The U2AF651,2L structures characterize ribose (r) nucleotides at all of the binding sites except the seventh and eighth deoxy-(d)U, which are likely to lack 2′-hydroxyl contacts based on the RNA-bound dU2AF651,2 structure. Qualitatively, a subset of the U2AF651,2L-nucleotide-binding sites (sites 1–3 and 7–9) share similar locations to those of the dU2AF651,2 structures (Supplementary Figs 2c,d and 3). Yet, only the U2AF651,2L interactions at sites 1 and 7 are nearly identical to those of the dU2AF651,2 structures (Supplementary Fig. 3a,f). In striking departures from prior partial views, the U2AF651,2L structures reveal three unanticipated nucleotide-binding sites at the centre of the Py tract, as well as numerous new interactions that underlie cognate recognition of the Py tract (Fig. 3a–h). site SO: melaniev@ebi.ac.uk 2023-03-20T16:50:02Z U2AF65-binding sites structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:03Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:50:08Z Py-tract nucleotides mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:13Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:50:12Z structures protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:50:22Z nucleotides evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:50:25Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:50:33Z RRM2 extension structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:30Z nucleotide-binding sites site SO: melaniev@ebi.ac.uk 2023-03-20T16:50:53Z RRM1/RRM2 junction structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:51:02Z RRM1 extension chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:39Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:51:33Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:51:37Z ribose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:51:40Z r chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:51:45Z nucleotides site SO: melaniev@ebi.ac.uk 2023-03-20T16:51:50Z binding sites residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:02Z seventh residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:08Z eighth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:52:16Z deoxy-(d)U protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:36Z structure site SO: melaniev@ebi.ac.uk 2023-03-20T16:52:45Z U2AF651,2L-nucleotide-binding sites site SO: melaniev@ebi.ac.uk 2023-03-20T16:52:50Z sites 1–3 site SO: melaniev@ebi.ac.uk 2023-03-20T16:52:54Z 7–9 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:58Z structures mutant MESH: melaniev@ebi.ac.uk 2023-06-15T11:53:56Z U2AF651,2L site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:06Z sites 1 and 7 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:53:10Z structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:53:18Z structures site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:30Z nucleotide-binding sites chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract RESULTS title_2 8134 U2AF65 inter-RRM linker interacts with the Py tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract RESULTS paragraph 8186 The U2AF651,2L RRM2, the inter-RRM linker and RRM1 concomitantly recognize the three central nucleotides of the Py tract, which are likely to coordinate the conformational arrangement of these disparate portions of the protein. Residues in the C-terminal region of the U2AF65 inter-RRM linker comprise a centrally located binding site for the fifth nucleotide on the RRM2 surface and abutting the RRM1/RRM2 interface (Fig. 3d). The backbone amide of the linker V254 and the carbonyl of T252 engage in hydrogen bonds with the rU5-O4 and -N3H atoms. In the C-terminal β-strand of RRM1, the side chains of K225 and R227 donate additional hydrogen bonds to the rU5-O2 lone pair electrons. The C-terminal region of the inter-RRM linker also participates in the preceding rU4-binding site, where the V254 backbone carbonyl and D256 carboxylate position the K260 side chain to hydrogen bond with the rU4-O4 (Fig. 3c). Otherwise, the rU4 nucleotide packs against F304 in the signature ribonucleoprotein consensus motif (RNP)-2 of RRM2. mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:57:34Z nucleotides chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:09Z Py tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:58:12Z C-terminal region protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:17Z binding site residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:25Z fifth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:36Z RRM2 surface site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:44Z RRM1/RRM2 interface structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:58:49Z linker residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:03Z T252 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:22Z rU5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:59:34Z β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:40Z K225 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:47Z R227 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:22Z rU5 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:58:12Z C-terminal region structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker site SO: melaniev@ebi.ac.uk 2023-03-20T17:00:00Z rU4-binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:00:11Z D256 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:00:19Z K260 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:00:39Z rU4 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:00:39Z rU4 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:01:06Z F304 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:01:15Z ribonucleoprotein consensus motif (RNP)-2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 RESULTS paragraph 9217 At the opposite side of the central fifth nucleotide, the sixth rU6 nucleotide is located at the inter-RRM1/RRM2 interface (Fig. 3e; Supplementary Movie 1). This nucleotide twists to face away from the U2AF65 linker and instead inserts the rU6-uracil into a sandwich between the β2/β3 loops of RRM1 and RRM2. The rU6 base edge is relatively solvent exposed; accordingly, the rU6 hydrogen bonds with U2AF65 are water mediated apart from a single direct interaction by the RRM1-N196 side chain. residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:25Z fifth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:25Z sixth residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:32Z rU6 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide site SO: melaniev@ebi.ac.uk 2023-03-20T17:03:40Z inter-RRM1/RRM2 interface chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:03:46Z linker residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:32Z rU6 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:06:59Z uracil structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:03:55Z β2/β3 loops structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:32Z rU6 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:59Z solvent exposed residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:32Z rU6 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:04:09Z water structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:04:19Z N196 RESULTS paragraph 9716 We tested the contribution of the U2AF651,2L interactions with the new central nucleotide to Py-tract affinity (Fig. 3i; Supplementary Fig. 4a,b). Mutagenesis of either V254 in the U2AF65 inter-RRM linker to proline or RRM1–R227 to alanine, which remove the hydrogen bond with the fifth uracil-O4 or -O2, reduced the affinities of U2AF651,2L for the representative AdML Py tract by four- or five-fold, respectively. The energetic penalties due to these mutations (ΔΔG 0.8–0.9 kcal mol−1) are consistent with the loss of each hydrogen bond with the rU5 base and support the relevance of the central nucleotide interactions observed in the U2AF651,2L structures. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T17:06:01Z tested the contribution mutant MESH: melaniev@ebi.ac.uk 2023-06-15T11:54:58Z U2AF651,2L chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:06:18Z Py-tract affinity experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T17:06:23Z Mutagenesis residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:06:33Z proline structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:47Z R227 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:06:44Z alanine residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:25Z fifth residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:07:00Z uracil evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:07:08Z affinities mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:15Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:07:36Z ΔΔG residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:22Z rU5 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:07:50Z structures RESULTS title_2 10389 U2AF65 RRM extensions interact with the Py tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract RESULTS paragraph 10438 The N- and C-terminal extensions of the U2AF65 RRM1 and RRM2 directly contact the bound Py tract. Rather than interacting with a new 5′-terminal nucleotide as we had hypothesized, the C-terminal α-helix of RRM2 instead folds across one surface of rU3 in the third binding site (Fig. 3b). There, a salt bridge between the K340 side chain and nucleotide phosphate, as well as G338-base stacking and a hydrogen bond between the backbone amide of G338 and the rU3-O4, secure the RRM2 extension. Indirectly, the additional contacts with the third nucleotide shift the rU2 nucleotide in the second binding site closer to the C-terminal β-strand of RRM2. Consequently, the U2AF651,2L-bound rU2-O4 and -N3H form dual hydrogen bonds with the K329 backbone atoms (Fig. 3a), rather than a single hydrogen bond with the K329 side chain as in the prior dU2AF651,2 structure (Supplementary Fig. 3b). structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:15:47Z N- and C-terminal extensions protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:15:55Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:16:11Z α-helix structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:16:19Z rU3 site SO: melaniev@ebi.ac.uk 2023-03-20T17:16:25Z third binding site residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:16:42Z K340 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:02Z G338 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:02Z G338 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:16:19Z rU3 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:50:33Z RRM2 extension residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:33Z third chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:50Z nucleotide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:58Z rU2 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:51Z nucleotide site SO: melaniev@ebi.ac.uk 2023-03-21T14:50:21Z second binding site structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:48:59Z β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:24Z U2AF651,2L-bound residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:58Z rU2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:18:12Z K329 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:18:12Z K329 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:18:20Z structure RESULTS paragraph 11331 At the N terminus, the α-helical extension of U2AF65 RRM1 positions the Q147 side chain to bridge the eighth and ninth nucleotides at the 3′ terminus of the Py tract (Fig. 3f–h). The Q147 residue participates in hydrogen bonds with the -N3H of the eighth uracil and -O2 of the ninth pyrimidine. The adjacent R146 guanidinium group donates hydrogen bonds to the 3′-terminal ribose-O2′ and O3′ atoms, where it could form a salt bridge with a phospho-diester group in the context of a longer pre-mRNA. Consistent with loss of a hydrogen bond with the ninth pyrimidine-O2 (ΔΔG 1.0 kcal mol−1), mutation of the Q147 to an alanine reduced U2AF651,2L affinity for the AdML Py tract by five-fold (Fig. 3i; Supplementary Fig. 4c). We compare U2AF65 interactions with uracil relative to cytosine pyrimidines at the ninth binding site in Fig. 3g,h and the Supplementary Discussion. structure_element SO: melaniev@ebi.ac.uk 2023-03-20T17:20:40Z α-helical extension protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:20:46Z Q147 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:08Z eighth residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:21:03Z ninth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:21:09Z nucleotides site SO: melaniev@ebi.ac.uk 2023-03-20T16:17:10Z 3′ terminus chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:20:46Z Q147 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:08Z eighth residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:07:00Z uracil residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:21:03Z ninth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:21:27Z pyrimidine residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:21:35Z R146 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:21:43Z ribose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:22:08Z pre-mRNA residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:21:03Z ninth chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:21:27Z pyrimidine evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:22:35Z ΔΔG experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T17:22:44Z mutation residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:20:46Z Q147 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:06:44Z alanine evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:08Z U2AF651,2L affinity gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T17:22:55Z compare protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:07:00Z uracil residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:23:03Z cytosine chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T17:23:11Z pyrimidines site SO: melaniev@ebi.ac.uk 2023-03-20T17:23:24Z ninth binding site RESULTS title_2 12221 Versatile primary sequence of the U2AF65 inter-RRM linker protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker RESULTS paragraph 12279 The U2AF651,2L structures reveal that the inter-RRM linker mediates an extensive interface with the second α-helix of RRM1, the β2/β3 strands of RRM2 and the N-terminal α-helical extension of RRM1. Altogether, the U2AF65 inter-RRM linker residues (R228–K260) bury 2,800 Å2 of surface area in the U2AF651,2L holo-protein, suggestive of a cognate interface compared with 1,900 Å2 for a typical protein–protein complex. The path of the linker initiates at P229 following the core RRM1 β-strand, in a kink that is positioned by intra-molecular stacking among the consecutive R228, Y232 and P234 side chains (Fig. 4a, lower right). A second kink at P236, coupled with respective packing of the L235 and M238 side chains on the N-terminal α-helical RRM1 extension and the core RRM1 α2-helix, reverses the direction of the inter-RRM linker towards the RRM1/RRM2 interface and away from the RNA-binding site. In the neighbouring apical region of the linker, the V244 and V246 side chains pack in a hydrophobic pocket between two α-helices of the core RRM1. The adjacent V249 and V250 are notable for their respective interactions that connect RRM1 and RRM2 at this distal interface from the RNA-binding site (Fig. 4a, top). A third kink stacks P247 and G248 with Y245 and re-orients the C-terminal region of the linker towards the RRM2 and bound RNA. At the RNA surface, the key V254 that recognizes the fifth uracil is secured via hydrophobic contacts between its side chain and the β-sheet surface of RRM2, chiefly the consensus RNP1-F304 residue that stacks with the fourth uracil (Fig. 4a, lower left). Few direct contacts are made between the remaining residues of the linker and the U2AF65 RRM2; instead, the C-terminal conformation of the linker appears primarily RNA mediated (Fig. 3c,d). mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:09:23Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker site SO: melaniev@ebi.ac.uk 2023-03-21T09:09:29Z extensive interface structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:09:52Z α-helix structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:09:56Z β2/β3 strands structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:10:00Z α-helical extension structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:10:07Z R228–K260 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:10:11Z holo-protein site SO: melaniev@ebi.ac.uk 2023-03-21T09:10:14Z cognate interface structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:10:19Z linker residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:10:31Z P229 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:10:35Z β-strand structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:10:39Z kink residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:10:52Z R228 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:02Z Y232 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:12Z P234 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:11:24Z second kink residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:35Z P236 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:45Z L235 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:53Z M238 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:12:01Z α-helical RRM1 extension protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:12:06Z α2-helix structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:44Z RRM1/RRM2 interface site SO: melaniev@ebi.ac.uk 2023-03-21T09:12:17Z RNA-binding site structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:12:36Z linker residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:46Z V244 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:54Z V246 site SO: melaniev@ebi.ac.uk 2023-03-21T09:13:01Z hydrophobic pocket structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:13:05Z α-helices protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:39Z V249 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:47Z V250 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 site SO: melaniev@ebi.ac.uk 2023-03-21T09:13:49Z interface site SO: melaniev@ebi.ac.uk 2023-03-21T09:12:17Z RNA-binding site structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:13:13Z third kink residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:13:25Z P247 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:13:32Z G248 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:13:39Z Y245 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:58:13Z C-terminal region structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:14:02Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:24Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:14:19Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:14:24Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:14:54Z RNA residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:25Z fifth residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:07:00Z uracil structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:15:11Z β-sheet surface structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:15:15Z RNP1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:01:06Z F304 residue_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:34Z fourth residue_name SO: melaniev@ebi.ac.uk 2023-03-20T17:07:00Z uracil structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:15:29Z linker protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:15:34Z linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:15:37Z RNA RESULTS paragraph 14100 We investigated whether the observed contacts between the RRMs and linker were critical for RNA binding by structure-guided mutagenesis (Fig. 4b). We titrated these mutant U2AF651,2L proteins into fluorescein-labelled AdML Py-tract RNA and fit the fluorescence anisotropy changes to obtain the apparent equilibrium affinities (Supplementary Fig. 4d–h). We introduced glycine substitutions to maximally reduce the buried surface area without directly interfering with its hydrogen bonds between backbone atoms and the base. First, we replaced V249 and V250 at the RRM1/RRM2 interface and V254 at the bound RNA site with glycine (3Gly). However, the resulting decrease in the AdML RNA affinity of the U2AF651,2L-3Gly mutant relative to wild-type protein was not significant (Fig. 4b). In parallel, we replaced five linker residues (S251, T252, V253, V254 and P255) at the fifth nucleotide-binding site with glycines (5Gly) and also found that the RNA affinity of the U2AF651,2L-5Gly mutant likewise decreased only slightly relative to wild-type protein. A more conservative substitution of these five residues (251–255) with an unrelated sequence capable of backbone-mediated hydrogen bonds (STVVP>NLALA) confirmed the subtle impact of this versatile inter-RRM sequence on affinity for the AdML Py tract. Finally, to ensure that these selective mutations were sufficient to disrupt the linker/RRM contacts, we substituted glycine for the majority of buried hydrophobic residues in the inter-RRM linker (including M144, L235, M238, V244, V246, V249, V250, S251, T252, V253, V254, P255; called 12Gly). Despite 12 concurrent mutations, the AdML RNA affinity of the U2AF651,2L-12Gly variant was reduced by only three-fold relative to the unmodified protein (Fig. 4b), which is less than the penalty of the V254P mutation that disrupts the rU5 hydrogen bond (Fig. 3d,i). structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:20:33Z linker experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:20:36Z structure-guided mutagenesis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:20:40Z titrated protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:29Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:20:44Z fluorescein gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:20:57Z Py-tract RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:02Z fluorescence anisotropy changes evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:07Z equilibrium affinities residue_name SO: melaniev@ebi.ac.uk 2023-03-21T09:21:18Z glycine experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:21:24Z substitutions experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:21:32Z replaced residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:39Z V249 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:47Z V250 site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:44Z RRM1/RRM2 interface residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:59Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:22:18Z RNA residue_name SO: melaniev@ebi.ac.uk 2023-03-21T09:21:18Z glycine mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:22:23Z 3Gly gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:26:32Z U2AF651,2L-3Gly protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:33Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-21T09:22:43Z protein experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:22:47Z replaced structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:22:50Z linker residues residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:56Z S251 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:04Z T252 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:06Z V253 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:21Z P255 site SO: melaniev@ebi.ac.uk 2023-03-21T09:24:25Z fifth nucleotide-binding site residue_name SO: melaniev@ebi.ac.uk 2023-03-21T09:24:41Z glycines mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:24:46Z 5Gly evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:26:35Z U2AF651,2L-5Gly protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:37Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-21T09:24:53Z protein experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:24:56Z conservative substitution residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:25:02Z 251–255 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:25:09Z STVVP>NLALA structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:25:13Z inter-RRM sequence evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:25:17Z affinity gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract structure_element SO: melaniev@ebi.ac.uk 2023-06-15T11:57:13Z linker structure_element SO: melaniev@ebi.ac.uk 2023-06-15T11:57:22Z RRM experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:37Z substituted residue_name SO: melaniev@ebi.ac.uk 2023-03-21T09:21:18Z glycine structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:25:36Z M144 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:45Z L235 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:53Z M238 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:46Z V244 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:54Z V246 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:39Z V249 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:47Z V250 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:56Z S251 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:04Z T252 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:06Z V253 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:21Z P255 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:26:06Z 12Gly experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:41Z 12 concurrent mutations gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:26:29Z U2AF651,2L-12Gly protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:48:40Z variant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:26:40Z unmodified protein PR: melaniev@ebi.ac.uk 2023-03-21T09:26:43Z protein mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:23Z rU5 RESULTS paragraph 15968 To test the interplay of the U2AF65 inter-RRM linker with its N- and C-terminal RRM extensions, we constructed an internal linker deletion of 20-residues within the extended RNA-binding domain (dU2AF651,2L). We found that the affinity of dU2AF651,2L for the AdML RNA was significantly reduced relative to U2AF651,2L (four-fold, Figs 1b and 4b; Supplementary Fig. 4i). Yet, it is well known that the linker deletion in the context of the minimal RRM1–RRM2 boundaries has no detectable effect on the RNA affinities of dU2AF651,2 compared with U2AF651,2 (refs; Figs 1b and 4b; Supplementary Fig. 4j). The U2AF651,2L structures suggest that an extended conformation of the truncated dU2AF651,2 inter-RRM linker would suffice to connect the U2AF651,2L RRM1 C terminus to the N terminus of RRM2 (24 Å distance between U2AF651,2L R227-Cα–H259-Cα atoms), which agrees with the greater RNA affinities of dU2AF651,2 and U2AF651,2 dual RRMs compared with the individual U2AF65 RRMs. However, stretching of the truncated dU2AF651,2L linker to connect the RRM termini is expected to disrupt its nucleotide interactions. Likewise, deletion of the N-terminal RRM1 extension in the shortened constructs would remove packing interactions that position the linker in a kinked turn following P229 (Fig. 4a), consistent with the lower RNA affinities of dU2AF651,2L, dU2AF651,2 and U2AF651,2 compared with U2AF651,2L. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:30:55Z constructed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T12:57:18Z linker deletion residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:31:18Z 20-residues protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:25Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:31:24Z RNA-binding domain mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:36Z affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:05Z RNA mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:31:39Z linker deletion protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:39Z RNA affinities mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:13Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:17Z extended conformation protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:20Z truncated mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:48Z R227 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:31Z H259 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:43Z RNA affinities mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:36Z dual structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:46Z individual protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:32:51Z truncated mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:32:54Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:26Z RRM termini experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:33:05Z deletion structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:51:02Z RRM1 extension protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:57Z shortened structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:33:24Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:33:27Z kinked turn residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:10:31Z P229 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:46Z RNA affinities mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L RESULTS paragraph 17374 To further test cooperation among the U2AF65 RRM extensions and inter-RRM linker for RNA recognition, we tested the impact of a triple Q147A/V254P/R227A mutation (U2AF651,2L-3Mut) for RNA binding (Fig. 4b; Supplementary Fig. 4d). Notably, the Q147A/V254P/R227A mutation reduced the RNA affinity of the U2AF651,2L-3Mut protein by 30-fold more than would be expected based on simple addition of the ΔΔG's for the single mutations. This difference indicates that the linearly distant regions of the U2AF65 primary sequence, including Q147 in the N-terminal RRM1 extension and R227/V254 in the N-/C-terminal linker regions at the fifth nucleotide site, cooperatively recognize the Py tract. Altogether, we conclude that the conformation of the U2AF65 inter-RRM linker is key for recognizing RNA and is positioned by the RRM extension but otherwise relatively independent of the side chain composition. The non-additive effects of the Q147A/V254P/R227A triple mutation, coupled with the context-dependent penalties of an internal U2AF65 linker deletion, highlights the importance of the structural interplay among the U2AF65 linker and the N- and C-terminal extensions flanking the core RRMs. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:38Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:35:53Z mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:35:56Z U2AF651,2L-3Mut mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:38Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:36:10Z mutation evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity mutant MESH: melaniev@ebi.ac.uk 2023-03-21T09:36:13Z U2AF651,2L-3Mut evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:22:38Z ΔΔG protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:36:16Z linearly distant structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:36:22Z regions protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:20:47Z Q147 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:51:02Z RRM1 extension residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:48Z R227 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:36:37Z linker regions site SO: melaniev@ebi.ac.uk 2023-03-21T09:36:41Z fifth nucleotide site chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:09Z RNA structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:30Z RRM extension mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:38Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:36:58Z triple mutation protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:27Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:37:02Z linker deletion protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:37:06Z linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:37:10Z N- and C-terminal extensions protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs RESULTS title_2 18570 Importance of U2AF65–RNA contacts for pre-mRNA splicing complex_assembly GO: melaniev@ebi.ac.uk 2023-06-15T12:02:00Z U2AF65–RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:51:29Z pre-mRNA RESULTS paragraph 18628 We proceeded to test the importance of new U2AF65–Py-tract interactions for splicing of a model pre-mRNA substrate in a human cell line (Fig. 5; Supplementary Fig. 5). As a representative splicing substrate, we utilized a well-characterized minigene splicing reporter (called pyPY) comprising a weak (that is, degenerate, py) and strong (that is, U-rich, PY) polypyrimidine tracts preceding two alternative splice sites (Fig. 5a). When transfected into HEK293T cells containing only endogenous U2AF65, the PY splice site is used and the remaining transcript remains unspliced. When co-transfected with an expression plasmid for wild-type U2AF65, use of the py splice site significantly increases (by more than five-fold) and as documented converts a fraction of the unspliced to spliced transcript. The strong PY splice site is insensitive to added U2AF65, suggesting that endogenous U2AF65 levels are sufficient to saturate this site (Supplementary Fig. 5b). We introduced the triple mutation (V254P/R227A/Q147A) that significantly reduced U2AF651,2L association with the Py tract (Fig. 4b) in the context of full-length U2AF65 (U2AF65-3Mut). Co-transfection of the U2AF65-3Mut with the pyPY splicing substrate significantly reduced splicing of the weak ‘py' splice site relative to wild-type U2AF65 (Fig. 5b,c). We conclude that the Py-tract interactions with these residues of the U2AF65 inter-RRM linker and RRM extensions are important for splicing as well as for binding a representative of the major U2-class of splice sites. complex_assembly GO: melaniev@ebi.ac.uk 2023-06-15T12:02:17Z U2AF65–Py-tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:22Z pre-mRNA species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:29Z minigene splicing reporter chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:40Z pyPY chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:43Z py structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:35Z U-rich chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:31Z PY chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:15Z polypyrimidine tracts site SO: melaniev@ebi.ac.uk 2023-03-20T16:19:52Z splice sites experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:43:26Z transfected protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:42:07Z endogenous protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-21T14:50:25Z PY splice site experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:43:43Z co-transfected experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:43:46Z expression plasmid protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-21T09:43:57Z py splice site site SO: melaniev@ebi.ac.uk 2023-03-21T09:44:08Z PY splice site protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:42:07Z endogenous protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:44:12Z triple mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:38Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:55:01Z U2AF65-3Mut experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:44:34Z Co-transfection mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:55:01Z U2AF65-3Mut chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:40Z pyPY site SO: melaniev@ebi.ac.uk 2023-03-21T14:50:29Z ‘py' splice site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-06-15T11:59:05Z Py-tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-21T09:45:08Z major U2-class of splice sites RESULTS title_2 20165 Sparse inter-RRM contacts underlie apo-U2AF65 dynamics structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:02Z inter-RRM protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 RESULTS paragraph 20220 The direct interface between U2AF651,2L RRM1 and RRM2 is minor, burying 265 Å2 of solvent accessible surface area compared with 570 Å2 on average for a crystal packing interface. A handful of inter-RRM hydrogen bonds are apparent between the side chains of RRM1-N155 and RRM2-K292, RRM1-N155 and RRM2-D272 as well as the backbone atoms of RRM1-G221 and RRM2-D273 (Fig. 4c). This minor U2AF65 RRM1/RRM2 interface, coupled with the versatile sequence of the inter-RRM linker, highlighted the potential role for inter-RRM conformational dynamics in U2AF65-splice site recognition. Paramagnetic resonance enhancement (PRE) measurements previously had suggested a predominant back-to-back, or ‘closed' conformation of the apo-U2AF651,2 RRM1 and RRM2 in equilibrium with a minor ‘open' conformation resembling the RNA-bound inter-RRM arrangement. Yet, small-angle X-ray scattering (SAXS) data indicated that both the minimal U2AF651,2 and longer constructs comprise a highly diverse continuum of conformations in the absence of RNA that includes the ‘closed' and ‘open' conformations. To complement the static portraits of U2AF651,2L structure that we had determined by X-ray crystallography, we used smFRET to characterize the probability distribution functions and time dependence of U2AF65 inter-RRM conformational dynamics in solution. site SO: melaniev@ebi.ac.uk 2023-03-21T09:55:15Z interface mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:02Z inter-RRM structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:24Z N155 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:27Z K292 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:31Z N155 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:35Z D272 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:38Z G221 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:55:41Z D273 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:44Z RRM1/RRM2 interface structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:02Z inter-RRM protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:17Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:52:03Z Paramagnetic resonance enhancement experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:21Z PRE protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:42:12Z back-to-back protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:12Z open protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:02Z inter-RRM experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:56:33Z small-angle X-ray scattering experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:56:36Z SAXS protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:25Z minimal mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:45Z highly diverse continuum of conformations protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:13Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:12Z open mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:57:01Z structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T09:57:04Z X-ray crystallography experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:50:22Z probability distribution functions protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM RESULTS paragraph 21568 The inter-RRM dynamics of U2AF65 were followed using FRET between fluorophores attached to RRM1 and RRM2 (Fig. 6a,b, Methods). The positions of single cysteine mutations for fluorophore attachment (A181C in RRM1 and Q324C in RRM2) were chosen based on inspection of the U2AF651,2L structures and the ‘closed' model of apo-U2AF651,2. Criteria included (i) residue locations that are distant from and hence not expected to interfere with the RRM/RNA or inter-RRM interfaces, (ii) inter-dye distances (50 Å for U2AF651,2L–Py tract and 30 Å for the closed apo-model) that are expected to be near the Förster radius (Ro) for the Cy3/Cy5 pair (56 Å), where changes in the efficiency of energy transfer are most sensitive to distance, and (iii) FRET efficiencies that are calculated to be significantly greater for the ‘closed' apo-model as opposed to the ‘open' RNA-bound structures (by ∼30%). The FRET efficiencies of either of these structurally characterized conformations also are expected to be significantly greater than elongated U2AF65 conformations that lack inter-RRM contacts. structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T12:56:16Z FRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:56:19Z fluorophores structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 residue_name SO: melaniev@ebi.ac.uk 2023-03-21T12:56:22Z cysteine experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T12:57:03Z mutations chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:57:31Z fluorophore mutant MESH: melaniev@ebi.ac.uk 2023-03-21T12:56:30Z A181C structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T12:56:33Z Q324C structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:58:07Z structures protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T12:58:12Z RRM/RNA site SO: melaniev@ebi.ac.uk 2023-03-21T12:58:16Z inter-RRM interfaces complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T12:58:20Z U2AF651,2L–Py tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T12:58:28Z Förster radius (Ro) chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:59:01Z FRET efficiencies protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:12Z open protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:59:05Z structures evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:59:01Z FRET efficiencies protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:59:16Z elongated protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T12:59:19Z lack structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:02:44Z RRM RESULTS paragraph 22670 Double-cysteine variant of U2AF651,2 was modified with equimolar amount of Cy3 and Cy5. Only traces that showed single photobleaching events for both donor and acceptor dyes and anti-correlated changes in acceptor and donor fluorescence were included in smFRET data analysis. Hence, molecules that were conjugated to two donor or two acceptor fluorophores were excluded from analysis. residue_name SO: melaniev@ebi.ac.uk 2023-03-21T13:00:04Z cysteine protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:00:14Z variant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:00:43Z modified chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET RESULTS paragraph 23055 We first characterized the conformational dynamics spectrum of U2AF65 in the absence of RNA (Fig. 6c,d; Supplementary Fig. 7a,b). The double-labelled U2AF651,2LFRET(Cy3/Cy5) protein was tethered to a slide via biotin-NTA/Ni+2 resin. Virtually no fluorescent molecules were detected in the absence of biotin-NTA/Ni+2, which demonstrates the absence of detectable non-specific binding of U2AF651,2LFRET to the slide. The FRET distribution histogram built from more than a thousand traces of U2AF651,2LFRET(Cy3/Cy5) in the absence of ligand showed an extremely broad distribution centred at a FRET efficiency of ∼0.4 (Fig. 6d). Approximately 40% of the smFRET traces showed apparent transitions between multiple FRET values (for example, Fig. 6c). Despite the large width of the FRET-distribution histogram, the majority (80%) of traces that showed fluctuations sampled only two distinct FRET states (for example, Supplementary Fig. 7a). Approximately 70% of observed fluctuations were interchanges between the ∼0.65 and ∼0.45 FRET values (Supplementary Fig. 7b). We cannot exclude a possibility that tethering of U2AF651,2LFRET(Cy3/Cy5) to the microscope slide introduces structural heterogeneity into the protein and, thus, contributes to the breadth of the FRET distribution histogram. However, the presence of repetitive fluctuations between particular FRET values supports the hypothesis that RNA-free U2AF65 samples several distinct conformations. This result is consistent with the broad ensembles of extended solution conformations that best fit the SAXS data collected for U2AF651,2 as well as for a longer construct (residues 136–347). We conclude that weak contacts between the U2AF65 RRM1 and RRM2 permit dissociation of these RRMs in the absence of RNA. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:18Z RNA mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:40Z tethered chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:52:39Z biotin-NTA/Ni+2 resin protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:40:49Z biotin-NTA/Ni+2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:50Z FRET distribution histogram evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:06:07Z ligand evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:17Z FRET efficiency experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:39Z FRET values evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:05Z FRET-distribution histogram evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:41:05Z FRET states evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:41Z FRET values mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:53:57Z FRET distribution histogram evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:41Z FRET values protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:13Z RNA-free protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:26Z extended experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:05:26Z SAXS mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:32Z 136–347 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:05:36Z RNA RESULTS title_2 24827 U2AF65 conformational selection and induced fit by bound RNA protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:06:39Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:06:42Z RNA RESULTS paragraph 24888 We next used smFRET to probe the conformational selection of distinct inter-RRM arrangements following association of U2AF65 with the AdML Py-tract prototype. Addition of the AdML RNA to tethered U2AF651,2LFRET(Cy3/Cy5) selectively increases a fraction of molecules showing an ∼0.45 apparent FRET efficiency, suggesting that RNA binding stabilizes a single conformation, which corresponds to the 0.45 FRET state (Fig. 6e,f). To assess the possible contributions of RNA-free conformations of U2AF65 and/or structural heterogeneity introduced by tethering of U2AF651,2LFRET(Cy3/Cy5) to the slide to the observed distribution of FRET values, we reversed the immobilization scheme. We tethered the AdML RNA to the slide via a biotinylated oligonucleotide DNA handle and added U2AF651,2LFRET(Cy3/Cy5) in the absence of biotin-NTA resin (Fig. 6g,h; Supplementary Fig. 7c–g). A 0.45 FRET value was again predominant, indicating a similar RNA-bound conformation and structural dynamics for the untethered and tethered U2AF651,2LFRET(Cy3/Cy5). experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:08Z Py-tract gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:22Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:40Z tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:20Z FRET efficiency evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:11Z FRET state protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:10:54Z RNA-free protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:09:58Z tethering mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:10:01Z distribution of FRET values experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:10:12Z reversed the immobilization scheme protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:40Z tethered gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:26Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:30Z biotinylated oligonucleotide DNA experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:46Z added mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:10:34Z biotin-NTA resin evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:17Z FRET value protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:28Z untethered protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:40Z tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 RESULTS paragraph 25927 We examined the effect on U2AF651,2L conformations of purine interruptions that often occur in relatively degenerate human Py tracts. We introduced an rArA purine dinucleotide within a variant of the AdML Py tract (detailed in Methods). Insertion of adenine nucleotides decreased binding affinity of U2AF65 to RNA by approximately five-fold. Nevertheless, in the presence of saturating concentrations of rArA-interrupted RNA slide-tethered U2AF651,2LFRET(Cy3/Cy5) showed a prevalent ∼0.45 apparent FRET value (Fig. 6i,j), which was also predominant in the presence of continuous Py tract. Therefore, RRM1-to-RRM2 distance remains similar regardless of whether U2AF65 is bound to interrupted or continuous Py tract. mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:13:55Z purine interruptions species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:14:03Z Py tracts experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:14:07Z introduced chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:14:35Z rArA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:14:51Z purine dinucleotide gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:15:44Z Insertion chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:16:30Z adenine nucleotides evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:16:01Z binding affinity protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:16:26Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:16:16Z rArA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:34Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:16:21Z slide-tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:15Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:28Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract RESULTS paragraph 26644 The inter-fluorophore distances derived from the observed 0.45 FRET state agree with the distances between the α-carbon atoms of the respective residues in the crystal structures of U2AF651,2L bound to Py-tract oligonucleotides. It should be noted that inferring distances from FRET values is prone to significant error because of uncertainties in the determination of fluorophore orientation factor κ2 and Förster radius R0, the parameters used in distance calculations. Nevertheless, the predominant 0.45 FRET state in the presence of RNA agrees with the Py-tract-bound crystal structure of U2AF651,2L. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:18:15Z inter-fluorophore distances evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:17Z FRET state evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:43:11Z Py-tract oligonucleotides evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:41Z FRET values evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:41:32Z FRET state chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:18:27Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:18:11Z Py-tract-bound evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:29Z crystal structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:40Z U2AF651,2L RESULTS paragraph 27257 Importantly, the majority of traces (∼70%) of U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA lacked FRET fluctuations and predominately exhibited a ∼0.45 FRET value (for example, Fig. 6g). The remaining ∼30% of traces for U2AF651,2LFRET(Cy3/Cy5) bound to the slide-tethered RNA showed fluctuations between distinct FRET values. The majority of traces that show fluctuations began at high (0.65–0.8) FRET value and transitioned to a ∼0.45 FRET value (Supplementary Fig. 7c–g). Hidden Markov modelling analysis of smFRET traces suggests that RNA-bound U2AF651,2L can sample at least two other conformations corresponding to ∼0.7–0.8 and ∼0.3 FRET values in addition to the predominant conformation corresponding to the 0.45 FRET state. Although a compact conformation (or multiple conformations) of U2AF651,2L corresponding to ∼0.7–0.8 FRET values can bind RNA, on RNA binding, these compact conformations of U2AF651,2L transition into a more stable structural state that corresponds to ∼0.45 FRET value and is likely similar to the side-by-side inter-RRM-arrangement of the U2AF651,2L crystal structures. Thus, the sequence of structural rearrangements of U2AF65 observed in smFRET traces (Supplementary Fig. 7c–g) suggests that a ‘conformational selection' mechanism of Py-tract recognition (that is, RNA ligand stabilization of a pre-configured U2AF65 conformation) is complemented by ‘induced fit' (that is, RNA-induced rearrangement of the U2AF65 RRMs to achieve the final ‘side-by-side' conformation), as discussed below. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:22:56Z RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:22:59Z RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:41Z FRET values evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:21:47Z Hidden Markov modelling analysis experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:41Z FRET values evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:09:17Z FRET state mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:42Z FRET values chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:23:11Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:41:47Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:23:35Z compact mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:09Z Py-tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:23:45Z pre-configured protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side DISCUSS title_1 28824 Discussion DISCUSS paragraph 28835 The U2AF65 structures and analyses presented here represent a successful step towards defining a molecular map of the 3′ splice site. Several observations indicate that the numerous intramolecular contacts, here revealed among the inter-RRM linker and RRM1, RRM2, and the N-terminal RRM1 extension, synergistically coordinate U2AF65–Py-tract recognition. Truncation of U2AF65 to the core RRM1–RRM2 region reduces its RNA affinity by 100-fold. Likewise, deletion of 20 inter-RRM linker residues significantly reduces U2AF65–RNA binding only when introduced in the context of the longer U2AF651,2L construct comprising the RRM extensions, which in turn position the linker for RNA interactions. Notably, a triple mutation of three residues (V254P, Q147A and R227A) in the respective inter-RRM linker, N- and C-terminal extensions non-additively reduce RNA binding by 150-fold. Altogether, these data indicate that interactions among the U2AF65 RRM1/RRM2, inter-RRM linker, N-and C-terminal extensions are mutually inter-dependent for cognate Py-tract recognition. The implications of this finding for U2AF65 conservation and Py-tract recognition are detailed in the Supplementary Discussion. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:26:55Z structures evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:26:57Z analyses site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:51:02Z RRM1 extension protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:17Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:27:17Z Truncation protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:47Z core structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:42Z RRM1–RRM2 region evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:10Z RNA affinity experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:27:24Z deletion residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:27:31Z 20 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:27:50Z inter-RRM linker residues protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:17Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:42:03Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:27:55Z longer mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:27:59Z linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:42:13Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:28:06Z triple mutation mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:38Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:28:19Z N- and C-terminal extensions evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:02Z RNA binding protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:05Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:28:26Z N-and C-terminal extensions chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:28:47Z Py-tract protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:09Z Py-tract DISCUSS paragraph 30033 Recently, high-throughput sequencing studies have shown that somatic mutations in pre-mRNA splicing factors occur in the majority of patients with myelodysplastic syndrome (MDS). MDS-relevant mutations are common in the small U2AF subunit (U2AF35, or U2AF1), yet such mutations are rare in the large U2AF65 subunit (also called U2AF2)—possibly due to the selective versus nearly universal requirements of these factors for splicing. A confirmed somatic mutation of U2AF65 in patients with MDS, L187V, is located on a solvent-exposed surface of RRM1 that is distinct from the RNA interface (Fig. 7a). This L187 surface is oriented towards the N terminus of the U2AF651,2L construct, where it is expected to abut the U2AF35-binding site in the context of the full-length U2AF heterodimer. Likewise, an unconfirmed M144I mutation reported by the same group corresponds to the N-terminal residue of U2AF651,2L, which is separated by only ∼20 residues from the U2AF35-binding site. As such, we suggest that the MDS-relevant U2AF65 mutations contribute to MDS progression indirectly, by destabilizing a relevant conformation of the conjoined U2AF35 subunit rather than affecting U2AF65 functions in RNA binding or spliceosome recruitment per se. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:31:12Z high-throughput sequencing studies protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:31:15Z pre-mRNA splicing factors protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:31:19Z small protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:31:27Z U2AF subunit protein PR: melaniev@ebi.ac.uk 2023-03-21T13:31:33Z U2AF35 protein PR: melaniev@ebi.ac.uk 2023-03-21T13:31:30Z U2AF1 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:31:38Z large protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-21T13:31:41Z U2AF2 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:31:46Z L187V site SO: melaniev@ebi.ac.uk 2023-03-21T13:31:55Z solvent-exposed surface structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 site SO: melaniev@ebi.ac.uk 2023-03-21T13:31:58Z RNA interface residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:31:50Z L187 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L site SO: melaniev@ebi.ac.uk 2023-03-21T13:32:03Z U2AF35-binding site protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-21T13:32:08Z U2AF oligomeric_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:55:53Z heterodimer mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:32:11Z M144I mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L site SO: melaniev@ebi.ac.uk 2023-03-21T13:32:14Z U2AF35-binding site protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-21T14:50:38Z U2AF35 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:42:28Z RNA complex_assembly GO: melaniev@ebi.ac.uk 2023-03-20T16:26:09Z spliceosome DISCUSS paragraph 31277 Our smFRET results agree with prior NMR/PRE evidence for multi-domain conformational selection as one mechanistic basis for U2AF65–RNA association (Fig. 7b). The ‘induced fit' versus ‘conformational selection' models are the prevailing views of the mechanisms underlying bio-molecular interactions (reviewed in ref.). In the former, ligand binding promotes a subsequent conformational change in the protein, whereas in the latter, the ligand selects a protein conformation from a pre-existing ensemble and thereby shifts the population towards that state. An ∼0.45 FRET value is likely to correspond to the U2AF65 conformation visualized in our U2AF651,2L crystal structures, in which the RRM1 and RRM2 bind side-by-side to the Py-tract oligonucleotide. The lesser 0.65–0.8 and 0.2–0.3 FRET values in the untethered U2AF651,2LFRET(Cy3/Cy5) experiment could correspond to respective variants of the ‘closed', back-to-back U2AF65 conformations characterized by NMR/PRE data, or to extended U2AF65 conformations, in which the intramolecular RRM1/RRM2 interactions have dissociated the protein is bound to RNA via single RRMs. An increased prevalence of the ∼0.45 FRET value following U2AF65–RNA binding, coupled with the apparent absence of transitions in many ∼0.45-value single molecule traces (for example, Fig. 6e), suggests a population shift in which RNA binds to (and draws the equilibrium towards) a pre-configured inter-RRM proximity that most often corresponds to the ∼0.45 FRET value. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:41:44Z NMR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:21Z PRE protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:17Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:42:40Z RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:19Z FRET value protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T13:41:59Z Py-tract oligonucleotide evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:03:42Z FRET values protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:28Z untethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:35Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:42:12Z back-to-back protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:50Z NMR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:21Z PRE protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:26Z extended protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:03:37Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:03:45Z RRM2 protein PR: melaniev@ebi.ac.uk 2023-03-21T13:42:32Z protein protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:39Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:42:29Z single structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:18Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:42:50Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:44Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:42:54Z pre-configured structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value DISCUSS paragraph 32792 Notably, our smFRET results reveal that U2AF65–Py-tract recognition can be characterized by an ‘extended conformational selection' model (Fig. 7b). In this recent model for macromolecular interactions, the pure ‘conformational selection' and ‘induced fit' scenarios represent the limits of a mechanistic spectrum and may compete or occur sequentially. Examples of ‘extended conformational selection' during ligand binding have been characterized for a growing number of macromolecules (for example, adenylate kinase, LAO-binding protein, poly-ubiquitin, maltose-binding protein and the preQ1 riboswitch, among others). Here, the majority of changes in smFRET traces for U2AF651,2LFRET(Cy3/Cy5) bound to slide-tethered RNA began at high (0.65–0.8) FRET value and transition to the predominant 0.45 FRET value (Supplementary Fig. 7c–g). These transitions could correspond to rearrangement from the ‘closed' NMR/PRE-based U2AF65 conformation in which the RNA-binding surface of only a single RRM is exposed and available for RNA binding, to the structural state seen in the side-by-side, RNA-bound crystal structure. As such, the smFRET approach reconciles prior inconsistencies between two major conformations that were detected by NMR/PRE experiments and a broad ensemble of diverse inter-RRM arrangements that fit the SAXS data for the apo-protein. Similar interdisciplinary structural approaches are likely to illuminate whether similar mechanistic bases for RNA binding are widespread among other members of the vast multi-RRM family. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET protein PR: melaniev@ebi.ac.uk 2023-03-21T13:53:18Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:43:04Z Py-tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:26Z extended protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:13Z adenylate kinase protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:16Z LAO-binding protein protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:19Z poly-ubiquitin protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:21Z maltose-binding protein protein_type MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:24Z preQ1 riboswitch experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:43Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:48Z RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:37Z NMR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:21Z PRE protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-21T14:50:34Z RNA-binding surface protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:45:31Z single structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:47Z RRM protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:29Z crystal structure experimental_method MESH: melaniev@ebi.ac.uk 2023-03-20T16:31:24Z smFRET experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:40Z NMR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:21Z PRE structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:45:45Z SAXS protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo protein PR: melaniev@ebi.ac.uk 2023-03-21T13:45:48Z protein structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:51Z RRM DISCUSS paragraph 34345 The finding that U2AF65 recognizes a nine base pair Py tract contributes to an elusive ‘code' for predicting splicing patterns from primary sequences in the post-genomic era (reviewed in ref.). Based on (i) similar RNA affinities of U2AF65 and U2AF651,2L, (ii) indistinguishable conformations among four U2AF651,2L structures in two different crystal packing arrangements and (iii) penalties of structure-guided mutations in RNA binding and splicing assays, we suggest that the extended inter-RRM regions of the U2AF651,2L structures underlie cognate Py-tract recognition by the full-length U2AF65 protein. Further research will be needed to understand the roles of SF1 and U2AF35 subunits in the conformational equilibria underlying U2AF65 association with Py tracts. Moreover, structural differences among U2AF65 homologues and paralogues may regulate splice site selection. Ultimately, these guidelines will assist the identification of 3′ splice sites and the relationship of disease-causing mutations to penalties for U2AF65 association. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:06Z RNA affinities protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:29Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:48:06Z structures evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:48:09Z penalties experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:48:11Z structure-guided mutations experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:48:16Z RNA binding and splicing assays protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:26Z extended structure_element SO: melaniev@ebi.ac.uk 2023-03-21T13:47:43Z inter-RRM regions mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:48:20Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:25:09Z Py-tract protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:30Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-21T13:48:25Z SF1 protein PR: melaniev@ebi.ac.uk 2023-03-21T13:48:29Z U2AF35 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:30Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:53Z Py tracts protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:30Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-20T16:21:04Z splice site site SO: melaniev@ebi.ac.uk 2023-03-20T16:12:54Z 3′ splice sites protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:30Z U2AF65 METHODS title_1 35392 Methods METHODS title_2 35400 Protein expression and purification METHODS paragraph 35436 For crystallization and RNA-binding experiments, human U2AF651,2L (residues 141–342 of NCBI RefSeq NP_009210) was expressed in Escherichia coli strain BL21 Rosetta-2 as a GST-fusion protein in the vector pGEX6P-2 and purified by glutathione affinity, followed by anion exchange and gel filtration chromatography. The GST-tagged protein was bound to a GSTrap column (GE Healthcare) in 1 M NaCl, 25 mM HEPES, pH 7.4 and eluted using 150 mM NaCl, 100 mM Tris, pH 8 containing 10 mM glutathione. The GST tag was cleaved from the protein by treatment with PreScission Protease during dialysis against a buffer containing 100 mM NaCl, 25 mM HEPES, pH 8, 5% (v/v) glycerol, 5 mM DTT, 0.25 mM EDTA and 0.1 mM PMSF. Cleaved GST was separated from the U2AF651,2L by subtractive glutathione affinity chromatography in 100 mM NaCl, 25 mM Tris, pH 8, 0.2 mM TCEP followed by subtractive anion-exchange chromatography with a HiTrap Q column (GE Healthcare). The final purification step was size-exclusion chromatography on a Superdex-75 prep-grade column (GE Healthcare) that had been previously equilibrated with 100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM tris(2-carboxy-ethyl)phosphine (TCEP). The purified U2AF651,2L was concentrated using a Vivaspin 15 R (Sartorius) centrifugal concentrator with 10 kDa MWCO, and the protein concentration was estimated using the calculated extinction coefficient of 8,940 M−1cm−1 and absorbance at 280 nm. Shorter constructs (U2AF651,2, residues 148–336; dU2AF651,2, residues 148–237, 258–336; dU2AF651,2L, residues 141–237, 258–342) (Fig. 1a) and individual U2AF651,2L Q147A, R227A, V254P mutants used for RNA-binding experiments were purified similarly. METHODS paragraph 37171 For comparative RNA-binding experiments, full-length human U2AF65 (residues 1–475) and the U2AF35-UHM (U2AF homology motif; residues 43–146, NCBI RefSeq NP_006749) initially were expressed and purified separately as GST fusion proteins. Following GST cleavage and ion-exchange chromatography (SP-HiTrap and Q-HiTrap, respectively), U2AF65 was combined with slight excess U2AF35-UHM (in stoichiometric ratio of 1:1.2) and dialysed overnight. The final U2AF heterodimer was purified by size-exclusion chromatography using a Superdex-200 prep-grade column (GE Healthcare) pre-equilibrated with 150 mM NaCl, 25 mM HEPES, pH 6.8, 0.2 mM TCEP. Representative purified U2AF651,2L and U2AF65–U2AF35-UHM proteins are shown in Supplementary Fig. 1a. METHODS title_2 37923 Oligonucleotide preparation METHODS paragraph 37951 High-performance liquid chromatography-purified oligonucleotides (sequences shown in Supplementary Fig. 2a) were purchased for crystallization (Integrated DNA Technologies, Inc.). The lyophilized oligonucleotides were diluted in gel filtration buffer for crystallization experiments. The 5′-fluorescein (Fl)-labelled RNAs (AdML: 5′-Fl-CCCUUUUUUUUCC-3′, Py tract of the AdML splicing substrate; 5′-4rU: 5′-Fl-CCUUUUCCCCCCC-3′; 3′-4rU: 5′-Fl-CCCCCCCUUUUCC-3′) for RNA-binding experiments (Dharmacon Research, Inc., Thermo Scientific) was deprotected according to the manufacturer's protocol, vacuum dried and resuspended in nuclease-free water. RNA and RNA–DNA concentrations were calculated using the calculated molar extinction coefficients and absorbance at 260 nm. METHODS title_2 38740 Fluorescence anisotropy RNA-binding experiments METHODS paragraph 38788 For RNA-binding experiments, purified proteins and RNA were diluted separately >100-fold in binding buffer (100 mM NaCl, 15 mM HEPES, pH 6.8, 0.2 mM TCEP, 0.1 U μl−1 Superase-In (Ambion Life Technologies)). The final RNA concentration in the cuvette was 30 nM. Volume changes during addition of the protein were <10% to minimize dilution effects. The fluorescence anisotropy changes during titration were measured using a FluoroMax-3 spectrophotometer temperature controlled by a circulating water bath at 23 °C. Samples were excited at 490 nm and emission intensities recorded at 520 nm with a slit width of 5 nm. The titrations were repeated three times in succession. Each titration was fit with Graphpad Prism v4.0 to obtain the apparent equilibrium dissociation constant (KD). The apparent equilibrium affinities (KA) are the reciprocal of the KD. The average KD's or KA's and s.e.m. among the three replicates were calculated using Excel and are reported in Figs 3 and 4; Supplementary Figs 1 and 4. The P values from a two-tailed unpaired t-test were calculated using Graphpad Prism v4.0. METHODS title_2 39908 Transfection, immunoblotting and RT-PCR analyses METHODS paragraph 39957 For transfection experiments, the full-length human U2AF65 cDNA in pCMV6-XL5 (Origene Tech. Inc., clone ID BC008740) was used (WT U2AF65) and in parallel mutated to encode the Q147A/R227A/V254P triple-mutant protein (Mut U2AF65). The pyPY minigene was a gift from M. Carmo-Fonseca (University of Lisbon, Portugal). HEK293T cells (kindly provided by Dr Lata Balakrishnan, originally purchased from ATCC, cat. no. CRL3216) were seeded into 12-well plates (2–4 × 105 cells per well) and grown as monolayers in MEM (Gibco Life Technologies) supplemented with 10% (v/v) of heat-inactivated fetal bovine serum, 1% (v/v) L-glutamine and 1% (v/v) penicillin–streptomycin. After 1 day, the cells were transiently transfected with either 0.5 μg of pyPY plasmid or a mixture of 0.5 μg of U2AF65 variant and 0.5 μg of pyPY plasmid per well using appropriately adjusted Lipofectamine 2000 (Invitrogen Life Technologies) ratio according to the manufacturer's instructions. METHODS paragraph 40932 For immunoblots of WT U2AF65 and Mut U2AF65 expression levels (Supplementary Fig. 5a), transfected or control cells were lysed in radioimmunoprecipitation assay buffer with proteinase and kinase inhibitors. Total protein (20 μg) was separated by SDS–PAGE, and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA) and immunoblotted using mouse monoclonal antibodies directed against U2AF65 (ref.) (MC3, cat. no. U4758 Sigma-Aldrich at 1:500 dilution) or as a control for comparison, GAPDH (glyceraldehyde-3-phosphate dehydrogenase; monoclonal clone 71.1, cat. no. G8795 Sigma-Aldrich at 1:5,000 dilution). Immunoblots were developed using anti-mouse horseradish peroxidase-conjugates (cat. no. U4758 Sigma-Aldrich, Co. at 1:2,500 or 1:10,000 dilutions for GAPDH and U2AF65, respectively) and detected using SuperSignal WestPico chemi-luminescent substrate (Pierce Thermo Scientific Inc.). Blots were imaged using a IS4000MM system (Carestream, Rochester, NY, USA). For size analysis, fluorescent images of the BioRad Precision Plus Dual Color Standards were overlaid directly. METHODS paragraph 42051 For reverse transcription PCR (RT-PCR), the total RNA was isolated 2 days post transfection using the Cells-to-cDNA II kit (Ambion Life Technologies). The RT-PCR reaction comprised 35 cycles (94 °C per 60 s—60 °C per 50 s—72 °C per 60 s) with forward (5′-TGAGGGGAGGTGAATGAGGAG-3′) and reverse (5′-TCCACTGGAAAGACCGCGAAG-3′) primers for the pyPY product or forward (5′-CATGTTCGTCATGGGTGTGAACCA-3′) and reverse (5′-ATGGCATGGACTGTGGTCATGAGT-3′) primers for a GAPDH control. The RT-PCR products were separated by 2% agarose gel electrophoresis and stained with ethidium bromide. The percentages of splice site use were calculated from the background corrected intensities I using the formula 100% × I(py)/[I(py)+I(PY)+I(unspliced)] for py spliced (Fig. 5b,c) or 100% × I(PY)/[I(py)+I(PY)+I(unspliced)] for PY spliced (Supplementary Fig. 5b). The band intensities of four independent biological replicates were measured using ImageQuant software. METHODS title_2 43029 Crystallization, data collection and structure determination METHODS paragraph 43090 Before crystallization, the purified U2AF651,2L and given oligonucleotide were mixed to achieve respective final concentrations of 1.0 and 1.1 mM and incubated on ice for 20–30 min. For each oligonucleotide, sparse matrix screens of the Jancarik and Kim Crystal Screen(in hanging drop format; Hampton Research, Corp.) and JCSG-Plus (in sitting drop format; Molecular Dimensions) were used to identify initial crystallization conditions, which were obtained from the latter screen and further optimized in hanging drop format. In optimized crystallization experiments, a mixture of sample and reservoir solution (1.2:1 μl) was equilibrated against 700 μl reservoir solution at 4 °C. METHODS paragraph 43787 The oligonucleotide sequences were optimized and the structures were determined as follows: in addition to the previously characterized dU2AF651,2-binding sites for seven nucleotides, the new terminal residues of the U2AF651,2L construct were presumed to contact an additional nucleotide and the crystal packing of a central nucleotide between the RRM1/RRM2 of dU2AF651,2 was presumed to represent one nucleotide. Also considering the known proclivity for deoxy(d)U to co-crystallize with dU2AF651,2 (ref.) and for 5-bromo-dU (5BrdU) to bind a given site of dU2AF651,2 (ref.), we initially designed two 9-mer oligonucleotides (5′-ribose (r)UrUrUrUrU(5BrdU)dUrUrU and 5′-rUrUrUdUdU(5BrdU)dUrUrU) and screened for co-crystallization with U2AF651,2L. The former oligonucleotide failed to produce crystals in these screens. The latter oligonucleotide comprising central dU nucleotides produced diffracting crystals, which were frozen directly from a reservoir comprising 100 mM phosphate–citrate buffer pH 4.2, 40% Peg 300. The structure determined by molecular replacement using Phenix with a data set collected at beamline (BL) 12-2 of the Stanford Synchrotron Radiation Lightsource (SSRL; Menlo Park, CA, USA) (Table 1). The search models comprising each of the individual RRMs bound to two nucleotides were derived from the dU2AF651,2 structure (PDB ID 2G4B) (translation function Z-score equivalent 12.9, log-likelihood gain 528). For comparison, searches with the NMR structure (PDB ID 2YH1) as a search model failed to find a solution. The initial structure revealed a greater number of central nucleotide-binding sites than expected. The oligonucleotide binding register had slipped to place the BrdU in the preferred site, leave the 5′ terminal-binding sites empty, and the terminal nucleotide unbound and disordered. Subsequent oligonucleotides were designed to place BrdU in the preferred site, fill the unoccupied 5′ terminal sites, capture rU at the central sites, and compare rC at the terminal site. METHODS paragraph 45810 The U2AF651,2L protein co-crystals with oligonucleotide 5′-phosphorylated (P)-rUrUdUdUrUdU(BrdU)dU were obtained using a reservoir of 200 mM LiCl, 100 mM sodium citrate pH 4.0, 8% (w/v) polyethylene glycol (PEG) 6,000, 10% (v/v) PEG 300, 10% (v/v) dioxane with 0.1 μl of N,N-bis[3-(D-gluconamido)propyl]deoxy-cholamide (deoxy-BigCHAP) (14 mM) added to the hanging drop and cryoprotected by sequential layering with reservoir solution supplemented with increasing PEG 300 to a final concentration of 26%. Co-crystals with either 5′-(P)rUrUdUrUrU(BrdU)dUdU or 5′-(P)rUrUrUdUrUrU(BrdU)dUrC were obtained from 1 M succinate, 100 mM HEPES, pH 7.0, 1–3% (w/v) PEG monomethylether 2,000. The former was cryoprotected by coating with a 1:1 (v/v) mixture of silicon oil and Paratone-N and the latter by sequential transfer to 21% (v/v) glycerol. Data sets for flash-cooled crystals were collected at 100 K using remote access to SSRL BL12-2. Structures were determined by molecular replacement using the initial U2AF651,2L/rUrUrUdUdU(BrdU)dUrUrU structure as a search model. Consistent sets of free-R reflections were maintained (6% of the total reflections). Models were built using COOT and refined with PHENIX. No non-glycine/non-proline residues were found in the disallowed regions of the Ramachandran plots. Clash scores and Molprobity scores calculated using the program Molprobity were above average. Structure illustrations were prepared using PYMOL. Crystallographic data and refinement statistics are given in Table 1. METHODS title_2 47354 Sample preparation for single-molecule FRET METHODS paragraph 47398 The U2AF651,2LFRET construct used for smFRET comprises the six histidine and T7 tags from the pET28a vector (Merck), a GGGS linker and U2AF65 residues 113–343. The single cysteine of human U2AF65 was replaced by alanine (C305A), which is a natural amino-acid variation among U2AF65 homologues. Single A181C and Q324C mutations were introduced in each RRM for fluorophore attachment at residues that were carefully selected to meet experimental criteria described in the Results. The U2AF651,2LFRET was purified by the same method as described above for U2AF651,2L protein and binds RNA with similar affinity as U2AF651,2L (Supplementary Fig. 6a). Before labelling, the purified U2AF651,2LFRET protein was incubated with 10 mM DTT on ice for 30 min and then buffer exchanged into Labelling Buffer (100 mM NaCl, 25 mM HEPES pH 7.0, 5 mM EDTA, 0.5 mM tris(2-carboxy-ethyl)phosphine (TCEP)) using Zeba Spin Desalting Columns 7K MWCO (Pierce, ThermoFisher Scientific). To initiate the labelling reaction, 4 μl each of cyanine (Cy)3-Maleimide and Cy5-Maleimide (Combinix, Inc.) stock solutions (10 mM in DMSO) were pre-mixed (total volume 8 μl) and then added to 200 μl of 20 μM protein (final 20:1 molar ratio of dye:protein). The labelling reaction was incubated at room temperature in the dark for 2 h and then quenched by the addition of 10 mM DTT. The labelled protein was separated from excess dye using a Zeba Spin Desalting Column followed by size exclusion chromatography using a pre-packed Superdex-75 10/300 GL (GE Healthcare) column in Labelling Buffer. Our previous experience of conjugating cysteines with maleimide derivatives of fluorophores and suggests that nonspecific modification of aminogroups of proteins with fluorescent dyes under the employed experimental conditions is negligible. Consistent with specific labelling of A181C and Q324C, the labelling efficiencies were ∼60% each for Cy3 and Cy5 as estimated using the dye extinction coefficients (ɛCy3=150,000 M−1 cm−1 at 550 nm, ɛCy5=170,000 M−1 cm−1 at 650 nm) and the calculated extinction coefficient of the U2AF651,2LFRET protein (ɛprot=8,940 M−1 cm−1 at 280 nm), and correcting for the absorbance (A) of the dyes at 280 nm (GE Healthcare, Amersham CyDye Maleimide product booklet): METHODS paragraph 49725 For smFRET experiments with a ‘strong', homogeneous Py tract, we used the prototypical AdML sequence (5′-CCUUUUUUUUCC-3′). To investigate the inter-RRM separation in the presence of a ‘weak' Py tract interrupted by purines, we compared the U2AF651,2L affinity for a purine-interrupted Py tract comprising an rUrUrUrUrU tract that is expected to bind U2AF65 RRM2/inter-RRM linker, a central rArA and an rUrUrUrCrC tract that is expected to bind RRM1. The tandem purines represent a compromise between significant inhibition of U2AF65 binding by longer A interruptions and an approximately five-fold penalty for the rArA mutation in the AdML Py tract (Supplementary Fig. 6b,c). To maintain avidity and provide flanking phosphoryl groups in case of inter-RRM adjustment, we included the 5′-C and 3′-A of parent AdML sequence, which are respective low-affinity nucleotides for binding RRM2 and RRM1 (ref.), in the final rArA-interrupted RNA oligonucleotide (5′-rCrUrUrUrUrUrArArUrUrUrCrCrA-3′). METHODS paragraph 50732 For the reversed immobilization of RNA via a complementary biotinyl-DNA primer experiment, the AdML Py-tract RNA was extended to include the DNA counterpart of downstream AdML intron/exon sequences that were complementary to the biotinyl-DNA primer. To increase separation from the slide surface, a hexaethylene glycol linker (18PEG) was inserted between the AdML Py-tract RNA and the tethered DNA duplex. The tethered oligonucleotide sequences included: 5′-rCrCrUrUrUrUrUrUrUrUrCrC/18PEG/dAdCdAdGdCdTdCdGdCdG-dGdTdTdGdAdGdGdAdCdAdA-3′ annealed to 5′-biotinyl-dTdTdGdTdCdCdTdCdAdA-dCdCdGdCdGdAdGdCdTdGdT-3' (purchased with high-performance liquid chromatography purification from Integrated DNA Technologies). METHODS title_2 51448 Single-molecule FRET data acquisition and analysis METHODS paragraph 51499 The smFRET measurements were carried out at room temperature in 50 mM HEPES, pH 7.4, 100 mM NaCl. The imaging buffer also contained an oxygen-scavenging system (0.8 mg ml−1 glucose oxidase, 0.625% glucose, 0.02 mg ml−1 catalase), 1.5 mM Trolox (used to eliminate Cy5 blinking) and 6 mM β-mercaptoethanol. The sample chamber was assembled from quartz microscope slides and glass cover slips coated with a mixture of m-PEG and biotin-PEG and pre-treated with neutravidin (0.2 mg ml−1). Surface tethering of doubly labelled U2AF651,2LFRET(Cy3/Cy5) via its His-tag (Fig. 6c–f,i,j; Supplementary Fig. 7a,b) was achieved by pre-incubating the sample chamber with 50 nM biotinyl-NTA resin (Biotin-X NTA, Biotium), pre-loaded with three-fold excess NiSO4) for 20 min before addition of 5 nM U2AF651,2LFRET(Cy3/Cy5). After 10 min, unbound sample was removed by washing the sample chamber with imaging buffer. The AdML RNA ligand was added to the imaging buffer at a concentration of 5 μM (100-fold higher than the measured KD value), whereas the rArA-interrupted RNA was added at a concentration of 10 μM. Alternatively, to detect binding of doubly labelled U2AF651,2LFRET(Cy3/Cy5) to surface-tethered RNA ligand (Fig. 6g,h Supplementary Fig. 7c–g), 10 nM AdML RNA (pre-annealed to biotinyl-DNA primer) was incubated in the neutravidin-treated sample chamber for 20 min, and 1 nM U2AF651,2LFRET(Cy3/Cy5) was then added to the imaging buffer. METHODS paragraph 52983 Single-molecule FRET measurements were taken as previously described. An Olympus IX71 inverted microscope, equipped with a UPlanApo 60x/1.20w objective lens, a 532 nm laser (Spectra-Physics) for excitation of Cy3 dyes, and a 642 nm laser (Spectra-Physics) for excitation of Cy5 dyes was used. Total internal reflection (TIR) was obtained by a quartz prism (ESKMA Optics). Fluorescence emission was split into Cy3 and Cy5 fluorescence using a dual view imaging system DV2 (Photometrics) equipped with a 630 nm dichroic mirror and recorded via an Andor iXon+ EMCCD camera. Movies were recorded using the Single software (downloaded from Prof. Taekjip Ha's laboratory website at the University of Illinois at Urbana-Champaign, physics.illinois.edu/cplc/software), with the exposure time set at 100 ms. We typically took up to five 5-minute-long movies while imaging different sections of the slide for each sample. Before each measurement, we checked for non-specific binding by adding doubly-labeled U2Fret to the slide in the absence of neutravidin and imaging the slide. Non-specific binding was virtually absent. METHODS paragraph 54105 Collected data sets were processed with IDL and Matlab softwares, using scripts downloaded from a freely available source: physics.illinois.edu/cplc/software. Apparent FRET efficiencies (Eapp) were calculated from the emission intensities of donor (ICy3) and acceptor (ICy5) as follows: Eapp=ICy5/(ICy5+ICy3). The FRET distribution histograms were built from traces that showed single-step photobleaching in both Cy3 and Cy5 signals using a Matlab script generously provided by Prof. Peter Cornish (University of Missouri, Columbia). Anti-correlated changes in donor and acceptor intensities with constant sum of intensities indicated the presence of an energy transfer in single molecules labelled with one donor and one acceptor dye. All histograms were smoothed with a five-point window and plotted using Origin software (Origin Lab Co). Idealization of FRET trajectories was done using the hidden Markov model algorithms via HaMMy software (http://bio.physics.illinois.edu/HaMMy.asp). Transition density plots were generated from transitions detected in idealized FRET trajectories obtained by HaMMy fit of raw FRET traces via Matlab. Frequency of transitions from starting FRET efficiency value (x-axis) to ending FRET efficiency value (y-axis) was represented by a heat map. The range of FRET efficiencies from 0 to 1 was separated in 200 bins. The resulting heat map was normalized to the most populated bin in the plot; the lower- and upper-bound thresholds were set to 20% and 100% of the most populated bin, respectively. METHODS paragraph 55637 The surface contour plots were generated as follows: the individual single-molecule FRET traces (for example, Fig. 6g of the main text and Supplementary Fig. 7e,f) were post synchronized at the first time point showing non-zero (>0.15) FRET efficiency, corresponding to binding. The time range (x-axis, 0–10 s) was separated into 100 bins. The FRET efficiency range (y-axis, 0–1 FRET) was separated into 100 bins. A heat map is used to represent the frequency of sampling of each FRET state over time; frequency in each bin was normalized to the most populated bin in the plot with lower- and upper-bound thresholds set at 10% and 80% of the most populated bin, respectively. 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USA ref 111 2014 60950 Following movement of domain IV of elongation factor G during ribosomal translocation 1941 1951 surname:McKinney;given-names:S. A. surname:Joo;given-names:C. surname:Ha;given-names:T. 16766620 REF Biophys. J. ref 91 2006 61036 Analysis of single-molecule FRET trajectories using hidden Markov modeling SUPPL footnote 61111 Author contributions A.A.A. performed crystallization, refinement, molecular biology and most RNA-binding experiments. E.S. performed smFRET experiments. R.C. labelled protein and S.H. completed a subset of RNA-binding experiments. C.L.K. cryoprotected crystals, collected crystallographic data and built structures. J.L.J. performed molecular replacement and completed structure refinements. M.R.G. and C.L.K. conceived the study. C.L.K. and D.N.E. designed the experiments. C.L.K., D.N.E. and E.S. wrote the paper with input from J.L.J. and A.A.A. ncomms10950-f1.jpg f1 FIG fig_title_caption 61661 The intact U2AF65 RRM1/RRM2-containing domain and flanking residues are required for binding contiguous Py tracts. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:30:59Z intact protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:03Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:51:58Z Py tracts ncomms10950-f1.jpg f1 FIG fig_caption 61776 (a) Domain organization of full-length (fl) U2AF65 and constructs used for RNA binding and structural experiments. The N- and C-terminal residue numbers are indicated. An internal deletion (d, Δ) of residues 238–257 removes a portion of the inter-RRM linker from the dU2AF651,2 and dU2AF651,2L constructs. (b) Comparison of the apparent equilibrium affinities of various U2AF65 constructs for binding the prototypical AdML Py tract (5′-CCCUUUUUUUUCC-3′). The flU2AF65 protein includes a heterodimerization domain of the U2AF35 subunit to promote solubility and folding. The apparent equilibrium dissociation constants (KD) for binding the AdML 13mer are as follows: flU2AF65, 30±3 nM; U2AF651,2L, 35±6 nM; U2AF651,2, 3,600±300 nM. (c) Comparison of the RNA sequence specificities of flU2AF65 and U2AF651,2L constructs binding C-rich Py tracts with 4U's embedded in either the 5′- (light grey fill) or 3′- (dark grey fill) regions. The KD's for binding 5′-CCUUUUCCCCCCC-3′ are: flU2AF65, 41±2 nM; U2AF651,2L, 31±3 nM. The KD's for binding 5′-CCCCCCCUUUUCC-3′ are: flU2AF65, 414±12 nM; U2AF651,2L, 417±10 nM. Bar graphs are hatched to match the constructs shown in a. The average apparent equilibrium affinity (KA) and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; NS, not significant, P>0.05. The purified protein and average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 1. RRM, RNA recognition motif; RS, arginine-serine rich; UHM, U2AF homology motif; ULM, U2AF ligand motif. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:34:30Z full-length protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:03:48Z fl protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:43:52Z RNA mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:03:59Z d mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:04:03Z Δ residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:04:06Z 238–257 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:06Z inter-RRM linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:04:12Z equilibrium affinities protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:04:30Z 5′-CCCUUUUUUUUCC-3′ protein PR: melaniev@ebi.ac.uk 2023-03-21T14:05:17Z flU2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:49:57Z heterodimerization domain protein PR: melaniev@ebi.ac.uk 2023-03-21T14:50:42Z U2AF35 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:26Z equilibrium dissociation constants evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:32Z KD gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML protein PR: melaniev@ebi.ac.uk 2023-03-21T14:05:17Z flU2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:12Z RNA sequence specificities protein PR: melaniev@ebi.ac.uk 2023-03-21T14:05:17Z flU2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:41Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:50:00Z C-rich chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:52:02Z Py tracts evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:32Z KD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:04:49Z 5′-CCUUUUCCCCCCC-3′ protein PR: melaniev@ebi.ac.uk 2023-03-21T14:05:17Z flU2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:32Z KD chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:05:09Z 5′-CCCCCCCUUUUCC-3′ protein PR: melaniev@ebi.ac.uk 2023-03-21T14:05:17Z flU2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:14:07Z average apparent equilibrium affinity evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:14:16Z KA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:14:18Z average fitted fluorescence anisotropy RNA-binding curves structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:50:04Z RRM structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:23Z RNA recognition motif structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:39Z RS structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:27Z arginine-serine rich structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:43Z UHM structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:45Z U2AF homology motif structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:48Z ULM structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:14:50Z U2AF ligand motif ncomms10950-f2.jpg f2 FIG fig_title_caption 63443 Structures of U2AF651,2L recognizing a contiguous Py tract. evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:15:10Z Structures mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:39:03Z contiguous chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract ncomms10950-f2.jpg f2 FIG fig_caption 63503 (a) Alignment of oligonucleotide sequences that were co-crystallized in the indicated U2AF651,2L structures. The regions of RRM1, RRM2 and linker contacts are indicated above by respective black and blue arrows from N- to C-terminus. For clarity, we consistently number the U2AF651,2L nucleotide-binding sites from one to nine, although in some cases the co-crystallized oligonucleotide comprises eight nucleotides and as such leaves the first binding site empty. The prior dU2AF651,2 nucleotide-binding sites are given in parentheses (site 4' interacts with dU2AF65 RRM1 and RRM2 by crystallographic symmetry). Italics, disordered in the structure. (b) Stereo views of a ‘kicked' 2|Fo|−|Fc| electron density map contoured at 1σ for the inter-RRM linker, N- and C-terminal residues (blue) or bound oligonucleotide of a representative U2AF651,2L structure (structure iv, bound to 5′-(P)rUrUrUdUrUrU(BrdU)dUrC) (magenta). (c) Cartoon diagram of this structure. Crystallographic statistics are given in Table 1 and the overall conformations of U2AF651,2L and prior dU2AF651,2/U2AF651,2 structures are compared in Supplementary Fig. 2. BrdU, 5-bromo-deoxy-uridine; d, deoxy-ribose; P-, 5′-phosphorylation; r, ribose. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:55Z Alignment chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:52:07Z oligonucleotide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:52:58Z co-crystallized mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:17:15Z structures structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:50:09Z linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:30Z nucleotide-binding sites experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:17:22Z co-crystallized chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:17:35Z oligonucleotide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:17:43Z nucleotides site SO: melaniev@ebi.ac.uk 2023-03-21T14:17:47Z first binding site mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:30Z nucleotide-binding sites mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:55:13Z dU2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:17:53Z 2|Fo|−|Fc| electron density map structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:06Z inter-RRM linker chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:52:14Z oligonucleotide mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:18:17Z structures chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:20Z BrdU chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:22Z 5-bromo-deoxy-uridine chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:26Z d chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:28Z deoxy-ribose chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:31Z P- chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:37Z 5′-phosphorylation chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:40Z r chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:18:43Z ribose ncomms10950-f3.jpg f3 FIG fig_title_caption 64725 Representative views of the U2AF651,2L interactions with each new nucleotide of the bound Py tract. mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:16:51Z nucleotide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:19:16Z bound chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract ncomms10950-f3.jpg f3 FIG fig_caption 64825 New residues of the U2AF651,2L structures are coloured a darker shade of blue, apart from residues that were tested by site-directed mutagenesis, which are coloured yellow. The nucleotide-binding sites of the U2AF651,2L and prior dU2AF651,2 structure are compared in Supplementary Fig. 3a–h. The first and seventh U2AF651,2L-binding sites are unchanged from the prior dU2AF651,2–RNA structure and are portrayed in Supplementary Fig. 3a,f. The four U2AF651,2L structures are similar with the exception of pH-dependent variations at the ninth site that are detailed in Supplementary Fig. 3i,j. The representative U2AF651,2L structure shown has the highest resolution and/or ribose nucleotide at the given site: (a) rU2 of structure iv; (b) rU3 of structure iii; (c) rU4 of structure i; (d) rU5 of structure iii; (e) rU6 of structure ii; (f) dU8 of structure iii; (g) dU9 of structure iii; (h) rC9 of structure iv. (i) Bar graph of apparent equilibrium affinities (KA) of the wild type (blue) and the indicated mutant (yellow) U2AF651,2L proteins binding the AdML Py tract (5′-CCCUUUUUUUUCC-3′). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; R227A, 166±2 nM; V254P, 137±10 nM; Q147A, 171±21 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; *P<0.05; NS, not significant, P>0.05. The average fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4a–c. mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:21:31Z structures experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:21:34Z site-directed mutagenesis site SO: melaniev@ebi.ac.uk 2023-03-20T16:53:30Z nucleotide-binding sites mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:21:50Z structure site SO: melaniev@ebi.ac.uk 2023-03-21T14:21:47Z first and seventh U2AF651,2L-binding sites complex_assembly GO: melaniev@ebi.ac.uk 2023-03-21T14:21:57Z dU2AF651,2–RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:00Z structure mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:03Z structures site SO: melaniev@ebi.ac.uk 2023-03-21T14:22:09Z ninth site mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:05Z structure chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:22:12Z ribose nucleotide residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:17:58Z rU2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:16:19Z rU3 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:00:39Z rU4 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:23Z rU5 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T17:03:32Z rU6 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:28Z dU8 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:32Z dU9 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:35Z rC9 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:39Z equilibrium affinities evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:23:12Z KA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:42Z wild type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:45Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:44:35Z 5′-CCCUUUUUUUUCC-3′ evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:49Z equilibrium dissociation constants evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:32Z KD mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:52Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:54Z wild type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:22:57Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:39Z Q147A evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:23:09Z KA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:23:17Z RNA-binding curves ncomms10950-f4.jpg f4 FIG fig_title_caption 66426 The U2AF65 linker/RRM and inter-RRM interactions. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:04:16Z linker structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:04:25Z RRM structure_element SO: melaniev@ebi.ac.uk 2023-06-15T12:04:36Z RRM ncomms10950-f4.jpg f4 FIG fig_caption 66476 (a) Contacts of the U2AF65 inter-RRM linker with the RRMs. A semi-transparent space-filling surface is shown for the RRM1 (green) and RRM2 (light blue). Residues V249, V250, V254 (yellow) are mutated to V249G/V250G/V254G in the 3Gly mutant; residues S251, T252, V253, P255 (red) along with V254 are mutated to S251G/T252G/V253G/V254G/P255G in the 5Gly mutant or to S251N/T252L/V253A/V254L/P255A in the NLALA mutant; residues M144, L235, M238, V244, V246 (orange) along with V249, V250, S251, T252, V253, V254, P255 are mutated to M144G/L235G/M238G/V244G/V246G/V249G/ V250G/S251G/T252G/V253G/V254G/P255G in the 12Gly mutant. Other linker residues are coloured either dark blue for new residues in the U2AF651,2L structure or light blue for the remaining inter-RRM residues. The central panel shows an overall view with stick diagrams for mutated residues; boxed regions are expanded to show the C-terminal (bottom left) and central linker regions (top) at the inter-RRM interfaces, and N-terminal linker region contacts with RRM1 (bottom right). (b) Bar graph of apparent equilibrium affinities (KA) for the AdML Py tract (5′-CCCUUUUUUUUCC-3′) of the wild-type (blue) U2AF651,2L protein compared with mutations of the residues shown in a: 3Gly (yellow), 5Gly (red), NLALA (hatched red), 12Gly (orange) and the linker deletions dU2AF651,2 in the minimal RRM1–RRM2 region (residues 148–237, 258–336) or dU2AF651,2L (residues 141–237, 258–342). The apparent equilibrium dissociation constants (KD) of the U2AF651,2L mutant proteins are: wild type (WT), 35±6 nM; 3Gly, 47±4 nM; 5Gly, 61±3 nM; 12Gly, 88±21 nM; NLALA, 45±3 nM; dU2AF651,2L, 123±5 nM; dU2AF651,2, 5000±100 nM; 3Mut, 5630±70 nM. The average KA and s.e.m. for three independent titrations are plotted. The P values from two-tailed unpaired t-tests with Welch's correction are indicated as follows: **P<0.01; *P<0.05; NS, not significant, P>0.05. The fitted fluorescence anisotropy RNA-binding curves are shown in Supplementary Fig. 4d–j. (c) Close view of the U2AF65 RRM1/RRM2 interface following a two-fold rotation about the x-axis relative to a. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:30:06Z inter-RRM linker structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:25Z RRM2 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:39Z V249 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:47Z V250 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:27:47Z mutated mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:27:53Z V249G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:27:59Z V250G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:06Z V254G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:08Z 3Gly mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:56Z S251 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:04Z T252 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:07Z V253 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:21Z P255 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:25Z mutated mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:33Z S251G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:40Z T252G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:47Z V253G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:06Z V254G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:57Z P255G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:00Z 5Gly mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:07Z S251N mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:15Z T252L mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:23Z V253A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:30Z V254L mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:36Z P255A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:29:39Z NLALA mutant residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:25:36Z M144 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:45Z L235 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:11:53Z M238 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:46Z V244 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:12:54Z V246 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:39Z V249 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:21:47Z V250 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:56Z S251 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:59:04Z T252 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:07Z V253 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:58:56Z V254 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:23:21Z P255 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:12Z mutated mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:20Z M144G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:28Z L235G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:34Z M238G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:42Z V244G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:30:49Z V246G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:27:53Z V249G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:27:59Z V250G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:33Z S251G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:40Z T252G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:47Z V253G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:06Z V254G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:28:57Z P255G mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:13Z 12Gly mutant structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:31:16Z linker mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-21T12:53:03Z inter-RRM structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:31:24Z central linker regions structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:31:27Z inter-RRM interfaces structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:31:33Z equilibrium affinities evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:31:36Z KA gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:44:52Z 5′-CCCUUUUUUUUCC-3′ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:47Z 3Gly mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:49Z 5Gly mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:51Z NLALA mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:54Z 12Gly experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:31:56Z linker deletions mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:14Z dU2AF651,2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:35:26Z minimal structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:50:16Z RRM1–RRM2 region residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:13Z 148–237 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:16Z 258–336 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:18Z 141–237 residue_range DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:21Z 258–342 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:11Z equilibrium dissociation constants evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:05:32Z KD mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:26Z mutant protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:29Z wild type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:31Z WT mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:32:38Z 3Gly mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:32:41Z 5Gly mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:32:44Z 12Gly mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:32:47Z NLALA mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:54:50Z dU2AF651,2L mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:15Z dU2AF651,2 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:32:51Z 3Mut evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:54Z KA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:32:57Z RNA-binding curves protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 site SO: melaniev@ebi.ac.uk 2023-03-20T16:58:45Z RRM1/RRM2 interface ncomms10950-f5.jpg f5 FIG fig_title_caption 68622 U2AF65 inter-domain residues are important for splicing a representative pre-mRNA substrate in human cells. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:33:34Z pre-mRNA species MESH: melaniev@ebi.ac.uk 2023-03-20T16:18:14Z human ncomms10950-f5.jpg f5 FIG fig_caption 68730 (a) Schematic diagram of the pyPY reporter minigene construct comprising two alternative splice sites preceded by either the weak IgM Py tract (py) or the strong AdML Py tract (PY) (sequences inset). (b) Representative RT-PCR of pyPY transcripts from HEK293T cells co-transfected with constructs encoding the pyPY minigene and either wild-type (WT) U2AF65 or a triple U2AF65 mutant (3Mut) of Q147A, R227A and V254P residues. (c) A bar graph of the average percentage of the py-spliced mRNA relative to total detected pyPY transcripts (spliced and unspliced) for the corresponding gel lanes (black, no U2AF65 added; white, WT U2AF65; grey, 3Mut U2AF65). The average percentages and s.d.'s are given among four independent biological replicates. ****P<0.0001 for two-tailed unpaired t-test with Welch's correction. Protein overexpression and qRT-PCR results are shown in Supplementary Fig. 5. chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:41Z pyPY site SO: melaniev@ebi.ac.uk 2023-03-20T16:19:52Z splice sites chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:43Z py gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:32Z PY experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:00Z RT-PCR chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:41Z pyPY experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:12Z co-transfected chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:41Z pyPY protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:22:39Z wild-type protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:36:19Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:36:23Z mutant mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:25Z 3Mut mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:39Z Q147A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:46Z R227A mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:36:54Z V254P chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:42:43Z py chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:37:02Z mRNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T09:41:41Z pyPY protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:37:07Z WT protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:37:09Z 3Mut protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:35:37Z Protein overexpression experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:35:54Z qRT-PCR ncomms10950-f6.jpg f6 FIG fig_title_caption 69621 RNA binding stabilizes the side-by-side conformation of U2AF65 RRMs. protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs ncomms10950-f6.jpg f6 FIG fig_caption 69690 (a,b) Views of FRET pairs chosen to follow the relative movement of RRM1 and RRM2 on the crystal structure of ‘side-by-side' U2AF651,2L RRMs bound to a Py-tract oligonucleotide (a, representative structure iv) or ‘closed' NMR/PRE-based model of U2AF651,2 (b, PDB ID 2YH0) in identical orientations of RRM2. The U2AF651,2LFRET proteins were doubly labelled at A181C/Q324C such that a mixture of Cy3/Cy5 fluorophores are expected to be present at each site. (c–f,i,j) The U2AF651,2LFRET(Cy3/Cy5) protein was immobilized on the microscope slide via biotin-NTA/Ni+2 (orange line) on a neutravidin (black X)-biotin-PEG (orange triangle)-treated surface and imaged either in the absence of ligands (c,d), in the presence of 5 μM AdML Py-tract RNA (5′-CCUUUUUUUUCC-3′) (e,f), or in the presence of 10 μM adenosine-interrupted variant RNA (5′-CUUUUUAAUUUCCA-3′) (i,j). In g and h, the immobilization protocol was reversed. The untethered U2AF651,2LFRET(Cy3/Cy5) protein (1 nM) was added to AdML RNA–polyethylene-glycol-linker–DNA oligonucleotide (10 nM), which was immobilized on the microscope slide by annealing with a complementary biotinyl-DNA oligonucleotide (black vertical line). Typical single-molecule FRET traces (c,e,g,i) show fluorescence intensities from Cy3 (green) and Cy5 (red) and the calculated apparent FRET efficiency (blue). Additional traces for untethered, RNA-bound U2AF651,2LFRET(Cy3/Cy5) are shown in Supplementary Fig. 7c,d. Histograms (d,f,h,j) show the distribution of FRET values in RNA-free, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (d); AdML RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (f); AdML RNA-bound, untethered U2AF651,2LFRET(Cy3/Cy5) (h) and adenosine-interrupted RNA-bound, slide-tethered U2AF651,2LFRET(Cy3/Cy5) (j). N is the number of single-molecule traces compiled for each histogram. experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:40:56Z FRET structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:26Z RRM2 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:46:29Z crystal structure protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:41:02Z Py-tract oligonucleotide protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:53:03Z NMR experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:22Z PRE mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:30:38Z U2AF651,2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:26Z RRM2 mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:33Z U2AF651,2LFRET mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:55:18Z A181C mutant MESH: melaniev@ebi.ac.uk 2023-03-21T14:55:21Z Q324C chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:41:11Z fluorophores mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:46:00Z biotin-NTA/Ni+2 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:05:45Z absence of chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:42:22Z ligands gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:41:16Z Py-tract RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:45:20Z 5′-CCUUUUUUUUCC-3′ residue_name SO: melaniev@ebi.ac.uk 2023-03-22T10:58:03Z adenosine chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:52:19Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:45:50Z 5′-CUUUUUAAUUUCCA-3′ protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:28Z untethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:42:32Z RNA–polyethylene-glycol-linker–DNA oligonucleotide chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:42:36Z biotinyl-DNA oligonucleotide experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:42:40Z single-molecule FRET evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:17Z calculated apparent FRET efficiency evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:28Z untethered protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:21Z Histograms evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:54:28Z distribution of FRET values protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:42:49Z RNA-free protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:42:55Z slide-tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:43:03Z slide-tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 gene GENE: melaniev@ebi.ac.uk 2023-03-20T16:36:16Z AdML protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:28Z untethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:52:31Z RNA-bound protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:43:15Z slide-tethered mutant MESH: melaniev@ebi.ac.uk 2023-03-21T13:04:34Z U2AF651,2LFRET chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:36Z Cy3 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T12:58:44Z Cy5 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:04:09Z traces evidence DUMMY: melaniev@ebi.ac.uk 2023-03-22T10:46:20Z histogram ncomms10950-f7.jpg f7 FIG fig_title_caption 71544 Schematic models of U2AF65 recognizing the Py tract. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-20T16:14:10Z Py tract ncomms10950-f7.jpg f7 FIG fig_caption 71597 (a) Diagram of the U2AF65, SF1 and U2AF35 splicing factors bound to the consensus elements of the 3′ splice site. A surface representation of U2AF651,2L is shown bound to nine nucleotides (nt); the relative distances and juxtaposition of the branch point sequence (BPS) and consensus AG dinucleotide at the 3′ splice site are unknown. MDS-relevant mutated residues of U2AF65 are shown as yellow spheres (L187 and M144). (b) Following binding to the Py-tract RNA, a conformation corresponding to high FRET and consistent with the ‘closed', back-to-back apo-U2AF65 model resulting from PRE/NMR characterization (PDB ID 2YH0) often transitions to a conformation corresponding to ∼0.45 FRET value, which is consistent with ‘open', side-by-side RRMs such as the U2AF651,2L crystal structures. Alternatively, a conformation of U2AF65 corresponding to ∼0.45 FRET value can directly bind to RNA; RNA binding stabilizes the ‘open', side-by-side conformation and thus shifts the U2AF65 population towards the ∼0.45 FRET value. RRM1, green; RRM2, pale blue; RRM extensions/linker, blue. protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 protein PR: melaniev@ebi.ac.uk 2023-03-21T14:50:48Z SF1 protein PR: melaniev@ebi.ac.uk 2023-03-21T14:50:51Z U2AF35 protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:13:37Z bound to chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:46:27Z nucleotides site SO: melaniev@ebi.ac.uk 2023-03-20T16:20:05Z branch point sequence site SO: melaniev@ebi.ac.uk 2023-03-20T16:24:06Z BPS chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:46:43Z AG dinucleotide site SO: melaniev@ebi.ac.uk 2023-03-20T16:09:33Z 3′ splice site protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:46:35Z L187 residue_name_number DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:25:36Z M144 chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:52:25Z Py-tract RNA evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T14:46:41Z high FRET protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:20Z closed protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:42:12Z back-to-back protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:45:31Z apo protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T13:42:22Z PRE experimental_method MESH: melaniev@ebi.ac.uk 2023-03-21T14:46:49Z NMR evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:12Z open protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:16:37Z RRMs mutant MESH: melaniev@ebi.ac.uk 2023-03-20T16:35:42Z U2AF651,2L evidence DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:29:39Z crystal structures protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value chemical CHEBI: melaniev@ebi.ac.uk 2023-03-21T14:47:02Z RNA chemical CHEBI: melaniev@ebi.ac.uk 2023-03-22T10:46:56Z RNA protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-21T09:56:12Z open protein_state DUMMY: melaniev@ebi.ac.uk 2023-03-20T16:45:08Z side-by-side protein PR: melaniev@ebi.ac.uk 2023-03-20T16:12:31Z U2AF65 evidence DUMMY: melaniev@ebi.ac.uk 2023-03-21T13:11:20Z FRET value structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:16Z RRM1 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:29:26Z RRM2 structure_element SO: melaniev@ebi.ac.uk 2023-03-20T16:17:00Z RRM extensions structure_element SO: melaniev@ebi.ac.uk 2023-03-21T14:46:59Z linker t1.xml t1 TABLE table_title_caption 72689 Crystallographic data and refinement statistics*. t1.xml t1 TABLE table <?xml version="1.0" encoding="UTF-8"?> <table frame="hsides" rules="groups" border="1"><colgroup><col align="left"/><col align="center"/><col align="center"/><col align="center"/><col align="center"/></colgroup><thead valign="bottom"><tr><th align="left" valign="top" charoff="50"><bold>Structure</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold>^{<bold>65</bold>}<bold>1,2L with rUrUrUdUdU(BrdU)dUrUrU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold>^{<bold>65</bold>}<bold>1,2L with (P)rUrUdUdUrUdU(BrdU)dU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold>^{<bold>65</bold>}<bold>1,2L with (P)rUrUdUrUrU(BrdU)dUdU</bold></th><th align="center" valign="top" charoff="50"><bold>U2AF</bold>^{<bold>65</bold>}<bold>1,2L with (P)rUrUrUdUrUrU(BrdU)dUrC</bold></th></tr></thead><tbody valign="top"><tr><td align="left" valign="top" charoff="50"><italic>Data collection</italic></td><td align="center" valign="top" charoff="50">(i)</td><td align="center" valign="top" charoff="50">(ii)</td><td align="center" valign="top" charoff="50">(iii)</td><td align="center" valign="top" charoff="50">(iv)</td></tr><tr><td align="left" valign="top" charoff="50">Space group</td><td align="center" valign="top" charoff="50">C222₁</td><td align="center" valign="top" charoff="50">C222₁</td><td align="center" valign="top" charoff="50">P2₁2₁2₁</td><td align="center" valign="top" charoff="50">P2₁2₁2₁</td></tr><tr><td align="left" valign="top" charoff="50">Unit cell (Å) <italic>a,b,c</italic></td><td align="center" valign="top" charoff="50">62.1, 114.2, 59.4</td><td align="center" valign="top" charoff="50">61.9, 115.1, 59.5</td><td align="center" valign="top" charoff="50">43.4, 62.2, 77.4</td><td align="center" valign="top" charoff="50">43.5, 63.4, 77.7</td></tr><tr><td align="left" valign="top" charoff="50">Resolution limits (Å)</td><td align="center" valign="top" charoff="50">32.46–2.04</td><td align="center" valign="top" charoff="50">32.57–1.86</td><td align="center" valign="top" charoff="50">38.71–1.50</td><td align="center" valign="top" charoff="50">38.83–1.57</td></tr><tr><td align="left" valign="top" charoff="50">Completeness (%)</td><td align="center" valign="top" charoff="50">95.5 (78.3)</td><td align="center" valign="top" charoff="50">98.7 (95.9)</td><td align="center" valign="top" charoff="50">98.2 (69.8)</td><td align="center" valign="top" charoff="50">98.3 (71.7)</td></tr><tr><td align="left" valign="top" charoff="50">Redundancy</td><td align="center" valign="top" charoff="50">4.6 (4.1)</td><td align="center" valign="top" charoff="50">4.3 (4.2)</td><td align="center" valign="top" charoff="50">6.1 (3.0)</td><td align="center" valign="top" charoff="50">6.2 (3.1)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>I</italic>/<italic>σ</italic>(<italic>I</italic>)</td><td align="center" valign="top" charoff="50">21.2 (4.2)</td><td align="center" valign="top" charoff="50">24.6 (4.6)</td><td align="center" valign="top" charoff="50">38.0 (6.5)</td><td align="center" valign="top" charoff="50">42.9 (6.9)</td></tr><tr><td align="left" valign="top" charoff="50"><italic>R</italic>_sym (%)</td><td align="center" valign="top" charoff="50">3.9 (32.1)</td><td align="center" valign="top" charoff="50">3.9 (30.3)</td><td align="center" valign="top" charoff="50">2.4 (14.8)</td><td align="center" valign="top" charoff="50">2.2 (14.9)</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>Refinement</italic></td></tr><tr><td align="left" valign="top" charoff="50"> No. reflections (work/test)</td><td align="center" valign="top" charoff="50">12,124/1,055</td><td align="center" valign="top" charoff="50">17,870/1,456</td><td align="center" valign="top" charoff="50">31,802/1,996</td><td align="center" valign="top" charoff="50">28,162/2,000</td></tr><tr><td align="left" valign="top" charoff="50"> <italic>R</italic>_work/<italic>R</italic>_free (%)</td><td align="center" valign="top" charoff="50">17.3/22.8</td><td align="center" valign="top" charoff="50">15.1/18.8</td><td align="center" valign="top" charoff="50">15.3/18.6</td><td align="center" valign="top" charoff="50">15.4/17.6</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>No. atoms</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="center" valign="top" charoff="50">2,982</td><td align="center" valign="top" charoff="50">3,052</td><td align="center" valign="top" charoff="50">2,986</td><td align="center" valign="top" charoff="50">2,978</td></tr><tr><td align="left" valign="top" charoff="50"> Oligonucleotide</td><td align="char" valign="top" char="." charoff="50">214</td><td align="char" valign="top" char="." charoff="50">209</td><td align="char" valign="top" char="." charoff="50">198</td><td align="char" valign="top" char="." charoff="50">255</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="char" valign="top" char="." charoff="50">118</td><td align="char" valign="top" char="." charoff="50">203</td><td align="char" valign="top" char="." charoff="50">263</td><td align="char" valign="top" char="." charoff="50">177</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>Bond r.m.s.d.</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Bond lengths (Å)</td><td align="char" valign="top" char="." charoff="50">0.013</td><td align="char" valign="top" char="." charoff="50">0.010</td><td align="char" valign="top" char="." charoff="50">0.008</td><td align="char" valign="top" char="." charoff="50">0.009</td></tr><tr><td align="left" valign="top" charoff="50"> Bond angles (°)</td><td align="char" valign="top" char="." charoff="50">1.32</td><td align="char" valign="top" char="." charoff="50">1.1</td><td align="char" valign="top" char="." charoff="50">1.05</td><td align="char" valign="top" char="." charoff="50">1.05</td></tr><tr><td colspan="5" align="center" valign="top" charoff="50"><italic>&lt;B&gt; factors (Ų)</italic></td></tr><tr><td align="left" valign="top" charoff="50"> Protein</td><td align="char" valign="top" char="." charoff="50">46.4</td><td align="char" valign="top" char="." charoff="50">27.4</td><td align="char" valign="top" char="." charoff="50">26.3</td><td align="char" valign="top" char="." charoff="50">26.7</td></tr><tr><td align="left" valign="top" charoff="50"> Oligonucleotide</td><td align="char" valign="top" char="." charoff="50">61.8</td><td align="char" valign="top" char="." charoff="50">35.2</td><td align="char" valign="top" char="." charoff="50">24.5</td><td align="char" valign="top" char="." charoff="50">30.5</td></tr><tr><td align="left" valign="top" charoff="50"> Water</td><td align="char" valign="top" char="." charoff="50">45.2</td><td align="char" valign="top" char="." charoff="50">35.2</td><td align="char" valign="top" char="." charoff="50">30.7</td><td align="char" valign="top" char="." charoff="50">29.8</td></tr></tbody></table> 72739 Structure U2AF651,2L with rUrUrUdUdU(BrdU)dUrUrU U2AF651,2L with (P)rUrUdUdUrUdU(BrdU)dU U2AF651,2L with (P)rUrUdUrUrU(BrdU)dUdU U2AF651,2L with (P)rUrUrUdUrUrU(BrdU)dUrC Data collection (i) (ii) (iii) (iv) Space group C2221 C2221 P212121 P212121 Unit cell (Å) a,b,c 62.1, 114.2, 59.4 61.9, 115.1, 59.5 43.4, 62.2, 77.4 43.5, 63.4, 77.7 Resolution limits (Å) 32.46–2.04 32.57–1.86 38.71–1.50 38.83–1.57 Completeness (%) 95.5 (78.3) 98.7 (95.9) 98.2 (69.8) 98.3 (71.7) Redundancy 4.6 (4.1) 4.3 (4.2) 6.1 (3.0) 6.2 (3.1) I/σ(I) 21.2 (4.2) 24.6 (4.6) 38.0 (6.5) 42.9 (6.9) Rsym (%) 3.9 (32.1) 3.9 (30.3) 2.4 (14.8) 2.2 (14.9) Refinement  No. reflections (work/test) 12,124/1,055 17,870/1,456 31,802/1,996 28,162/2,000  Rwork/Rfree (%) 17.3/22.8 15.1/18.8 15.3/18.6 15.4/17.6 No. atoms  Protein 2,982 3,052 2,986 2,978  Oligonucleotide 214 209 198 255  Water 118 203 263 177 Bond r.m.s.d.  Bond lengths (Å) 0.013 0.010 0.008 0.009  Bond angles (°) 1.32 1.1 1.05 1.05 <B> factors (Å2)  Protein 46.4 27.4 26.3 26.7  Oligonucleotide 61.8 35.2 24.5 30.5  Water 45.2 35.2 30.7 29.8 t1.xml t1 TABLE table_footnote 73893 All available crystallographic data was used for refinement. t1.xml t1 TABLE table_footnote 73954 *A single crystal was used for each structure. Values from the highest resolution shell are given in parentheses: 2.15–2.04; 1.90–1.86; 1.53–1.50; 1.61–1.57.