In Vitro ICLIP-based Modeling Uncovers How the Splicing Factor U2AF2 Relies on Regulation by Cofactors

Overview

Journal Genome Res

Specialty Genetics

Date 2018 Apr 13

PMID 29643205

Citations 39

Authors

F X Reymond Sutandy

Stefanie Ebersberger

Lu Huang

Anke Busch

Maximilian Bach

Hyun-Seo Kang

Jorg Fallmann

Daniel Maticzka

Rolf Backofen

Peter F Stadler

Kathi Zarnack

Michael Sattler

Stefan Legewie

Julian Konig

Affiliations

Soon will be listed here.

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 -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 -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 -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.

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