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ICLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): synthetic gene Homo sapiens

PROVIDER: GSE99700 | GEO | 2018/02/01

REPOSITORIES: GEO

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iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

Project description:Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF65 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We established 'in vitro iCLIP' experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF65 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We quantitatively measure U2AF65 affinities at hundreds of binding sites, and compare in vitro and in vivo binding landscapes by mathematical modelling. We find that trans-acting RBPs extensively regulate U2AF65 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of intronic regions. Using machine learning, we identify novel trans-acting RBPs (including FUBP1, BRUNOL6 and PCBP1) that modulate U2AF65 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.

2020-03-05 | GSE126693 | GEO

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

2020-03-05 | GSE126692 | GEO

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors [in vivo iCLIP]

2018-02-01 | GSE99688 | GEO

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors [in vitro iCLIP]

2018-02-01 | GSE99698 | GEO

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors [in vitro iCLIP co-factors]

2018-02-01 | GSE99696 | GEO

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

Project description:iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

| PRJNA389317 | ENA

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

Project description:iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

| PRJNA523002 | ENA

iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

Project description:iCLIP-based modeling uncovers 3’ splice site definition: how U2AF65 specificity relies on regulation by co-factors

| PRJNA523001 | ENA

In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors.

Project description:Alternative splicing generates distinct mRNA isoforms and is crucial for proteome diversity in eukaryotes. The RNA-binding protein (RBP) U2AF2 is central to splicing decisions, as it recognizes 3' splice sites and recruits the spliceosome. We establish "in vitro iCLIP" experiments, in which recombinant RBPs are incubated with long transcripts, to study how U2AF2 recognizes RNA sequences and how this is modulated by trans-acting RBPs. We measure U2AF2 affinities at hundreds of binding sites and compare in vitro and in vivo binding landscapes by mathematical modeling. We find that trans-acting RBPs extensively regulate U2AF2 binding in vivo, including enhanced recruitment to 3' splice sites and clearance of introns. Using machine learning, we identify and experimentally validate novel trans-acting RBPs (including FUBP1, CELF6, and PCBP1) that modulate U2AF2 binding and affect splicing outcomes. Our study offers a blueprint for the high-throughput characterization of in vitro mRNP assembly and in vivo splicing regulation.

| S-EPMC5932610 | biostudies-literature

U2AF65 assemblies drive sequence-specific splice site recognition.

Project description:The essential splicing factor U2AF65 is known to help anchoring U2 snRNP at the branch site. Its C-terminal UHM domain interacts with ULM motifs of SF3b155, an U2 snRNP protein. Here, we report a cooperative binding of U2AF65 and the related protein CAPERα to the multi-ULM domain of SF3b155. In addition, we show that the RS domain of U2AF65 drives a liquid-liquid phase separation that is amplified by intronic RNA with repeated pyrimidine tracts. In cells, knockdown of either U2AF65 or CAPERα improves the inclusion of cassette exons that are preceded by such repeated pyrimidine-rich motifs. These results support a model in which liquid-like assemblies of U2AF65 and CAPERα on repetitive pyrimidine-rich RNA sequences are driven by their RS domains, and facilitate the recruitment of the multi-ULM domain of SF3b155. We anticipate that posttranslational modifications and proteins recruited in dynamical U2AF65 and CAPERα condensates may further contribute to the complex mechanisms leading to specific splice site choice that occurs in cells.

| S-EPMC6681011 | biostudies-literature

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