A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

Kielkopf, Clara L.; Ermolenko, Dmitri N.; Jenkins, Jermaine L.; Laird, Kholiswa M.; Feeney, Callen F.; Glasser, Eliezra; Warnasooriya, Chandani

doi:10.1101/2019.12.31.891358

Cited by 5 publications

(7 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Lastly, because these early lariat formation events are characterized by a strong PPT and strong U2AF binding ( Fig. 5F-H ), and binding of U2AF2 to a strong PPT can induce an active conformation of U2AF2 independent of U2AF1 (Warnasooriya et al, 2020), we suggest that at some introns U2AF2 binds to a PPT in a manner also independent of either a downstream or upstream 5’ SS – that is, by neither canonical exon nor intron definition. Indeed, U2AF2 along with co-factors can bind in extract to a substrate that includes a BP, PPT, and 3’ splice site AG but lacks any 5’ SS (e.g., Mackereth et al, 2011; Maul-Newby et al, 2021), and the association of U2AF2 with RNAP II (David et al, 2011) could allow rapid and efficient recognition of the PPT.…”

Section: Discussionmentioning

confidence: 88%

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

Zeng

Zeng²,

Fair

et al. 2021

Preprint

View full text Add to dashboard Cite

As splicing is intimately coupled with transcription, understanding splicing mechanisms requires an understanding of splicing timing, which is currently limited. Here, we developed CoLa-seq (co-transcriptional lariat sequencing), a genomic assay that reports splicing timing relative to transcription through analysis of nascent lariat intermediates. In human cells, we mapped 165,282 branch points and characterized splicing timing for over 70,000 introns. Splicing timing varies dramatically across introns, with regulated introns splicing later than constitutive introns. Machine learning-based modeling revealed genetic elements predictive of splicing timing, notably the polypyrimidine tract, intron length, and regional GC content, which illustrate the significance of the broader genomic context of an intron and the impact of co-transcriptional splicing. The importance of the splicing factor U2AF in early splicing rationalizes surprising observations that most introns can splice independent of exon definition. Together, these findings establish a critical framework for investigating the mechanisms and regulation of co-transcriptional splicing.HighlightsCoLa-seq enables cell-type specific, genome-wide branch point annotation with unprecedented efficiency.CoLa-seq captures co-transcriptional splicing for tens of thousands of introns and reveals splicing timing varies dramatically across introns.Modeling uncovers key genetic determinants of splicing timing, most notably regional GC content, intron length, and the polypyrimidine tract, the binding site for U2AF2.Early splicing precedes transcription of a downstream 5’ SS and in some cases accessibility of the upstream 3’ SS, precluding exon definition.

show abstract

Section: Discussionmentioning

confidence: 88%

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

Zeng

Zeng²,

Fair

et al. 2021

Preprint

View full text Add to dashboard Cite

show abstract

“…A recent study, assessing in vitro U2AF1 binding, questioned sequence-specific reductions in binding; U2AF1 S34F compared to WT showed little discrimination for the -3 nucleotide sequence, with similar affinity to any nucleotide except G 25 . Similarly, limited -3 sequence specificity was also observed in studies of the effects of S34F mutation on the open vs closed conformations of U2AF2 57 . Our integrative analysis unveiled an unexpectedly multifaceted relationship between mutant U2AF1 binding and splicing outcomes.…”

Section: Discussionmentioning

confidence: 63%

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Biancon

Joshi

Zimmer

et al. 2021

Preprint

View full text Add to dashboard Cite

Somatic mutations in splicing factors are of significant interest in myeloid malignancies and other cancers. U2AF1, together with U2AF2, is essential for 3’ splice site recognition. U2AF1 mutations result in aberrant splicing, but the molecular mechanism and the full spectrum of consequences on RNA biology have not been fully elucidated to date. We performed multi-omics profiling of in vivo RNA binding, splicing and turnover for U2AF1 S34F and Q157R mutants. We dissected specific binding signals of U2AF1 and U2AF2 and showed that U2AF1 mutations individually alter U2AF1-RNA binding, resulting in defective U2AF2 recruitment. We demonstrated a complex relationship between differential binding and splicing, expanding upon the currently accepted loss-of-binding model. Finally, we observed that U2AF1 mutations increase the formation of stress granules in both cell lines and primary acute myeloid leukemia samples. Our results uncover U2AF1 mutation-dependent pathogenic RNA mechanisms and provide the basis for developing targeted therapeutic strategies.

show abstract

“…As such, U2AF2 transcends a traditional classification of either a "specific" or "nonspecific" RNA binding protein. One means for U2AF2 to fulfill its multifaceted role is to rely on a modular architecture of tandem RRMs, which differ in uridine-specificity and switch between "open" and "closed" conformations in response to the RNA sequence (4,6,11). Prior studies of U2AF2 RRM1/RRM2 bound to noncognate RNAs reveal a variety of changes, ranging from subtle shifts of the side chains and protein backbone to nucleotide rotations and syn/anti-conformer flips (10,11).…”

Section: Discussionmentioning

confidence: 99%

“…Multiple partners work to enhance and regulate U2AF2, including U2AF1, SF1, SF3B1, and PUF60/RBM39, among others. Cancer-associated mutations, most commonly in U2AF1 and in some cases U2AF2, also influence 3´ splice site recognition (52)(53)(54) and the distribution of U2AF2 conformations (6). Resolving the influence of additional subunits and cancer-associated mutations on the U2AF2-RNA conformation remains an important direction for future research.…”

Section: Discussionmentioning

confidence: 99%

“…The U2AF2 protein recognizes the Py tract via two tandem RNA recognition motifs (RRM1 and RRM2). In the absence of RNA or in the presence of degenerate Py tracts comprising less than four consecutive uridines, U2AF2 adopts a "closed" conformation in which RRM1 is masked and only RRM2 is available for RNA binding (4)(5)(6). When bound to a longer uridine tract such as the 3´ splice site consensus, the U2AF2 RRMs have an "open", side-by-side conformation with both RRM1 and RRM2 contacting the RNA (4,7).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

Glasser

Maji

Biancon

et al. 2021

Preprint

Self Cite

View full text Add to dashboard Cite

The essential pre-mRNA splicing factor U2AF2 (also called U2AF65) identifies polypyrimidine (Py) tract signals of nascent transcripts, despite length and sequence variations. Previous studies have shown that the U2AF2 RNA recognition motifs (RRM1 and RRM2) preferentially bind uridine-rich RNAs. Nonetheless, the specificity of the RRM1/RRM2 interface for the central Py tract nucleotide has yet to be investigated. We addressed this question by determining crystal structures of U2AF2 bound to a cytidine, guanosine, or adenosine at the central position of the Py tract, and compared U2AF2-bound uridine structures. Local movements of the RNA site accommodated the different nucleotides, whereas the polypeptide backbone remained similar among the structures. Accordingly, molecular dynamics simulations revealed flexible conformations of the central, U2AF2-bound nucleotide. The RNA binding affinities and splicing efficiencies of structure-guided mutants demonstrated that U2AF2 tolerates nucleotide substitutions at the central position of the Py tract. Moreover, enhanced crosslinking and immunoprecipitation of endogenous U2AF2 in human erythroleukemia cells showed uridine-sensitive binding sites with lower sequence conservation at the central nucleotide positions of otherwise uridine-rich, U2AF2-bound splice sites. Altogether, these results highlight the importance of RNA flexibility for protein recognition and take a step towards relating splice site motifs to pre-mRNA splicing efficiencies.

show abstract

A Splice Site-Sensing Conformational Switch in U2AF2 is Modulated by U2AF1 and its Recurrent Myelodysplasia-Associated Mutation

Cited by 5 publications

References 42 publications

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

Profiling of Nascent Lariat Intermediates Reveals Key Genetic Determinants of the Timing of Human Co-transcriptional Splicing

Multi-omics profiling of U2AF1 mutants dissects pathogenic mechanisms affecting RNA granules in myeloid malignancies

Pre-mRNA Splicing Factor U2AF2 Recognizes Distinct Conformations of Nucleotide Variants at the Center of the pre-mRNA Splice Site Signal

Contact Info

Product

Resources

About