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

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE126693 | GEO | 2020/03/05

REPOSITORIES: GEO

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

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.

2018-02-01 | GSE99698 | 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 vitro iCLIP co-factors]

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

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Mechanisms for U2AF to define 3M-bM-^@M-2 splice sites and regulate alternative splicing in the human genome

Project description:The U2AF heterodimer has been well studied for its role in defining functional 3M-bM-^@M-^Y splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3M-bM-^@M-^Y splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3M-bM-^@M-^Y splice site associated with either the alternative exon to cause exon skipping or with the competing constitutive exon to induce exon inclusion. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states. Examination of U2AF heterodimer regulated splicing in Hela cells with CLIP-seq (U2AF65), paired-end RNA-seq (si-NC and si-U2AF65) and RASL-seq (respective three biological replicates of WT, si-NC, si-U2AF65, si-U2AF35, si-NC + pcDNA3.0, si-U2AF65 + pcDNA3.0, and si-U2AF65 + Flag-U2AF35)

2014-10-08 | E-GEOD-61603 | biostudies-arrayexpress

Transcriptome-wide analysis of U2AF2 RNA binding in a human hematopoietic cell line.

Project description: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. 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. Keywords: splicing, RNA binding, U2AF2, U2AF65, 3'SS, 3' splice site, eCLIP, polypyrimidine tract, RNA recognition motif, RRM, U-rich, Py-tract

2022-06-01 | GSE195669 | GEO

A global regulatory mechanism for activating an exon network required for neurogenesis

Project description:The vertebrate and neural-specific SR-related protein nSR100/SRRM4 regulates an extensive program of alternative splicing with critical roles in nervous system development. However, the mechanism by which nSR100 controls its target exons is poorly understood. We demonstrate that nSR100-dependent neural exons are associated with a unique configuration of intronic cis-elements that promote rapid switch-like regulation during neurogenesis. A key feature of this configuration is the insertion of specialized intronic enhancers between polypyrimidine tracts and acceptor sites that bind nSR100 to potently activate exon inclusion in neural cells, while weakening 3' splice site recognition and contributing to exon skipping in non-neural cells. nSR100 further operates by forming multiple interactions with early spliceosome components bound proximal to 3' splice sites. These multifaceted interactions achieve dominance over neural exon silencing mediated by the splicing regulator PTBP1. The results thus illuminate a widespread mechanism by which a critical neural exon network is activated during neurogenesis. RNA-Seq was used to obtain mRNA profiles of various N2A and 293T cell lines from human and mouse, respectively, to investigate the roles of nSR100, Ptbp1 and U2af65 in alternative splicing regulation. PAR-iCLIP and iCLIP experiments followed by high throughput sequencing were conducted to obtain RNA binding profiles of nSR100, PTBP1 and U2af65.

2014-09-11 | E-GEOD-57278 | biostudies-arrayexpress

Transcriptome-wide mapping of hnRNP C and U2AF65 using iCLIP

Project description:iCLIP experiment to assess the binding of the highly abundant nuclear RNA-binding protein hnRNP C and core splicing factor U2AF65 on a genomic scale. To investigate how both proteins compete for binding at a subset of sites, U2AF65 iCLIP experiments were performed from both HNRNPC knockdown and control HeLa cells.

2013-01-31 | E-MTAB-1371 | biostudies-arrayexpress

Mechanisms for U2AF to define 3′ splice sites and regulate alternative splicing in the human genome

Project description:The U2AF heterodimer has been well studied for its role in defining functional 3’ splice sites in pre-mRNA splicing, but many fundamental questions still remain unaddressed regarding the function of U2AF in mammalian genomes. Through genome-wide analysis of U2AF-RNA interactions, we report that U2AF has the capacity to directly define ~88% of functional 3’ splice sites in the human genome, but numerous U2AF binding events also occur in intronic locations. Mechanistic dissection reveals that upstream intronic binding events interfere with the immediate downstream 3’ splice site associated with either the alternative exon to cause exon skipping or with the competing constitutive exon to induce exon inclusion. We further demonstrate partial functional impairment with mutations in U2AF35, but not U2AF65, in regulated splicing. These findings reveal the genomic function and regulatory mechanism of U2AF in both normal and disease states.

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Poly(A) RNA sequencing of Silencing Defective 2 (SDE2) depletion in HeLa cells via siRNA

Project description:Pre-mRNA splicing is a complex and dynamic process that relies on the intricate coordination between a multitude of cis-elements and trans-acting factors. Here, we identify and characterize Silencing Defective 2 (SDE2) as a human RNA binding protein and trans-acting splicing-associated factor required for efficient mRNA processing. In this study, we use Poly(A) purified RNA sequencing to identify the alternative splicing profile in Hela cells following depletion of SDE2 via siRNA. Our data demonstrate that SDE2 depletion causes widespread changes in alternative splicing (AS), with increased intron retention being the most common event. These retained introns are significantly shorter, have a higher GC content, and overall maintain weaker 5' and 3' splice sites than other introns throughout the genome. Following the depletion of SDE2, increased intron retention causes catastrophic consequences for the cell, including defects in mitotic progression, global loss of protein translation, and ultimately, complete loss of cellular viability. Taken together, we define SDE2 as a previously uncharacterized RNA binding protein that functions to regulate pre-mRNA splicing and ensure cellular viability.

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