An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal

Agrawal, Anant A.; Salsi, Enea; Chatrikhi, Rakesh; Henderson, Steven T.; Jenkins, Jermaine L.; Green, Michael R.; Ermolenko, Dmitri N.; Kielkopf, Clara L.

doi:10.1038/ncomms10950

Cited by 62 publications

(111 citation statements)

References 49 publications

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“…Additional chemical shift perturbations in the NMR data also revealed stabilizing RNA contacts for a few residues preceding the first β1-strand in RRM1 (Fig. S6A), consistent with a recent crystal structure of an extended RRM1,2 protein bound to a strong Pytract (38). However, the NMR titration data also demonstrate that no additional contacts are observed for residues preceding this N-terminal region of RRM1.…”

Section: Resultssupporting

confidence: 88%

“…This is fully consistent with the NMR spectra, which show a single set of signals, indicating conformational averaging that is fast on the NMR chemical shift time scale (i.e., less than milliseconds). We do not observe conformational dynamics on time scales longer than 30 ms. Agrawal et al (38) described conformational dynamics of U2AF65 on the time scale of 5-30 s. Although the different types of spFRET measurements reveal different dynamic time scales, they consistently show more dynamics in the absence of RNA, and the open conformation of U2AF65 is stabilized in the presence of a strong Py-tract.…”

Section: U2af35 Enhances Py-tract Recognition By a Dynamic Populationmentioning

confidence: 99%

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Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Voithenberg

Sánchez-Rico

Kang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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An essential early step in the assembly of human spliceosomes onto pre-mRNA involves the recognition of regulatory RNA cis elements in the 3′ splice site by the U2 auxiliary factor (U2AF). The large (U2AF65) and small (U2AF35) subunits of the U2AF heterodimer contact the polypyrimidine tract (Py-tract) and the AG-dinucleotide, respectively. The tandem RNA recognition motif domains (RRM1,2) of U2AF65 adopt closed/inactive and open/active conformations in the free form and when bound to bona fide Py-tract RNA ligands. To investigate the molecular mechanism and dynamics of 3′ splice site recognition by U2AF65 and the role of U2AF35 in the U2AF heterodimer, we have combined single-pair FRET and NMR experiments. In the absence of RNA, the RRM1,2 domain arrangement is highly dynamic on a submillisecond time scale, switching between closed and open conformations. The addition of Py-tract RNA ligands with increasing binding affinity (strength) gradually shifts the equilibrium toward an open conformation. Notably, the protein-RNA complex is rigid in the presence of a strong Py-tract but exhibits internal motion with weak Py-tracts. Surprisingly, the presence of U2AF35, whose UHM domain interacts with U2AF65 RRM1, increases the population of the open arrangement of U2AF65 RRM1,2 in the absence and presence of a weak Py-tract. These data indicate that the U2AF heterodimer promotes spliceosome assembly by a dynamic population shift toward the open conformation of U2AF65 to facilitate the recognition of weak Py-tracts at the 3′ splice site. The structure and RNA binding of the heterodimer was unaffected by cancer-linked myelodysplastic syndrome mutants.uring gene expression, the removal of introns is essential for translation of mature mRNA. The splicing process involves a large number of splicing factors for the correct recognition of introns (1). Whereas U1 snRNP contacts the 5′ splice site, recognition of the 3′ splice site involves binding of SF1/BBP to the branch point sequence (BPS) (2-5) and binding of the U2 auxiliary factor (U2AF) heterodimer to the poly-pyrimidine-tract (Py-tract) that precedes the AG dinucleotide at the intron/exon junction. Binding of U2AF to the 3′ splice site during the early steps of spliceosome assembly recruits the U2 snRNP (6-9). The strength, i.e., splicing efficiency, of a 3′ splice site requires recognition of the BPS, Py-tract, and the AG dinucleotide. However, of these three RNA elements, the Py-tract exhibits the largest degree of variability, and thus, weak to strong splice sites are primarily classified depending on the composition of the Py-tract (7, 10).U2AF is a heterodimer consisting of a large (U2AF65) and a small (U2AF35) subunit. U2AF65 harbors two canonical RNA recognition motifs (RRM1,2) and an atypical C-terminal RRM domain, called the U2AF homology motif (UHM). U2AF35 has one RRM (which acts as an UHM), flanked N-and C-terminally by two CCCH-type zinc finger motifs, respectively (Fig. S1A) (9, 11, 12). The U2AF heterodimer is formed by recognition of a peptide motif, cal...

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Section: Resultssupporting

confidence: 88%

Section: U2af35 Enhances Py-tract Recognition By a Dynamic Populationmentioning

confidence: 99%

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Voithenberg

Sánchez-Rico

Kang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…A Q147A change could reduce the affinity of U2AF large subunit for the polypyrimidine tract by 5 folds and this region might coordinate with the C-terminal region of RRM2 and the inter-RRM linker region to affect the affinity and specificity of RNA binding [54]. This study corroborates our findings that residues in the region between ULM and RRM1 are important for 3ʹ SS recognition and alternative splicing in C. elegans [35,38].…”

Section: Discussionsupporting

confidence: 85%

“…Recently, a structure-function study of U2AF large subunit suggests that residues of this region (a.a. 141 to 147 in mammalian U2AF large subunit) [54] are important for high-affinity binding to the polypyrimidine tract. A Q147A change could reduce the affinity of U2AF large subunit for the polypyrimidine tract by 5 folds and this region might coordinate with the C-terminal region of RRM2 and the inter-RRM linker region to affect the affinity and specificity of RNA binding [54].…”

Section: Discussionmentioning

confidence: 99%

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

Zhou

Gao

et al. 2018

RNA Biology

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A key step in pre-mRNA splicing is the recognition of 3ʹ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3ʹ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

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“…The branched intron lariat preferentially forms over a range of~18-40 nucleotides from the 3 0 -splice-site junction (63,64), and the branchpoint adenosine typically is separated from the start of the Py tract by eight poorly conserved nucleotides (64). Yet the known RNA interactions of the structurally characterized SF1 and U2AF 65 domains encompass only three nucleotides after the branchpoint adenosine (65,66) and nine pyrimidine nucleotides (56,67), leaving approximately five intervening nucleotides unaccounted for. Based on available structures, the SF1 coiled coil could act as an inert ruler of the BPS-Py tract spacing.…”

Section: Uhmk1 Phosphorylation Slightly Reduces Sf1-u2afmentioning

confidence: 99%

SF1 Phosphorylation Enhances Specific Binding to U2AF 65 and Reduces Binding to 3′-Splice-Site RNA

Chatrikhi

Wang

Gupta

et al. 2016

Biophysical Journal

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Splicing factor 1 (SF1) recognizes 3 0 splice sites of the major class of introns as a ternary complex with U2AF 65 and U2AF 35 splicing factors. A conserved SPSP motif in a coiled-coil domain of SF1 is highly phosphorylated in proliferating human cells and is required for cell proliferation. The UHM kinase 1 (UHMK1), also called KIS, double-phosphorylates both serines of this SF1 motif. Here, we use isothermal titration calorimetry to demonstrate that UHMK1 phosphorylation of the SF1 SPSP motif slightly enhances specific binding of phospho-SF1 to its cognate U2AF 65 protein partner. Conversely, quantitative fluorescence anisotropy RNA binding assays and isothermal titration calorimetry experiments establish that double-SPSP phosphorylation reduces phospho-SF1 and phospho-SF1-U2AF 65 binding affinities for either optimal or suboptimal splice-site RNAs. Domain-substitution and mutagenesis experiments further demonstrate that arginines surrounding the phosphorylated SF1 loop are required for cooperative 3 0 splice site recognition by the SF1-U2AF 65 complex (where cooperativity is defined as a nonadditive increase in RNA binding by the protein complex relative to the individual proteins). In the context of local, intracellular concentrations, the subtle effects of SF1 phosphorylation on its associations with U2AF 65 and splice-site RNAs are likely to influence pre-mRNA splicing. However, considering roles for SF1 in pre-mRNA retention and transcriptional repression, as well as in splicing, future comprehensive investigations are needed to fully explain the requirement for SF1 SPSP phosphorylation in proliferating human cells.

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An extended U2AF65–RNA-binding domain recognizes the 3′ splice site signal

Cited by 62 publications

References 49 publications

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

Recognition of the 3′ splice site RNA by the U2AF heterodimer involves a dynamic population shift

RBM-5 modulates U2AF large subunit-dependent alternative splicing inC. elegans

SF1 Phosphorylation Enhances Specific Binding to U2AF 65 and Reduces Binding to 3′-Splice-Site RNA

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