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Genome Res. 2018 May;28(5):699-713. doi: 10.1101/gr.229757.117. Epub 2018 Apr 11.

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

Author information

1
Institute of Molecular Biology (IMB) gGmbH, 55128 Mainz, Germany.
2
Institute of Structural Biology, Helmholtz Center Munich, 85764 Neuherberg, Germany.
3
Biomolecular NMR and Center for Integrated Protein Science Munich at Department of Chemistry, Technical University of Munich, 85747 Garching, Germany.
4
Bioinformatics Group, Department of Computer Science and Interdisciplinary Center for Bioinformatics, University of Leipzig, 04107 Leipzig, Germany.
5
Bioinformatics Group, Department of Computer Science, University of Freiburg, 79110 Freiburg, Germany.
6
Centre for Biological Signalling Studies (BIOSS), University of Freiburg, 79104 Freiburg, Germany.
7
Buchmann Institute for Molecular Life Sciences (BMLS), Goethe University Frankfurt, 60438 Frankfurt a.M., Germany.
#
Contributed equally

Abstract

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.

PMID:
29643205
PMCID:
PMC5932610
DOI:
10.1101/gr.229757.117
[Indexed for MEDLINE]
Free PMC Article

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