RT Journal
A1 Sutandy, F.X. Reymond
A1 Ebersberger, Stefanie
A1 Huang, Lu
A1 Busch, Anke
A1 Bach, Maximilian
A1 Kang, Hyun-Seo
A1 Fallmann, Jörg
A1 Maticzka, Daniel
A1 Backofen, Rolf
A1 Stadler, Peter F.
A1 Zarnack, Kathi
A1 Sattler, Michael
A1 Legewie, 
Stefan
A1 König, Julian
T1 In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
JF Genome Research 
JO Genome Research 
YR 2018 
FD May 01 
VO 28 
IS 5 
SP 699 
OP 713 
DO 10.1101/gr.229757.117 
UL http://genome.cshlp.org/content/28/5/699.abstract 
AB 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.