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

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 recombin...

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Autores principales: Sutandy, F.X. Reymond, Ebersberger, Stefanie, Huang, Lu, Busch, Anke, Bach, Maximilian, Kang, Hyun-Seo, Fallmann, Jörg, Maticzka, Daniel, Backofen, Rolf, Stadler, Peter F., Zarnack, Kathi, Sattler, Michael, Legewie, Stefan, König, Julian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932610/
https://www.ncbi.nlm.nih.gov/pubmed/29643205
http://dx.doi.org/10.1101/gr.229757.117
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author Sutandy, F.X. Reymond
Ebersberger, Stefanie
Huang, Lu
Busch, Anke
Bach, Maximilian
Kang, Hyun-Seo
Fallmann, Jörg
Maticzka, Daniel
Backofen, Rolf
Stadler, Peter F.
Zarnack, Kathi
Sattler, Michael
Legewie, Stefan
König, Julian
author_facet Sutandy, F.X. Reymond
Ebersberger, Stefanie
Huang, Lu
Busch, Anke
Bach, Maximilian
Kang, Hyun-Seo
Fallmann, Jörg
Maticzka, Daniel
Backofen, Rolf
Stadler, Peter F.
Zarnack, Kathi
Sattler, Michael
Legewie, Stefan
König, Julian
author_sort Sutandy, F.X. Reymond
collection PubMed
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.
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spelling pubmed-59326102018-11-01 In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors Sutandy, F.X. Reymond Ebersberger, Stefanie Huang, Lu Busch, Anke Bach, Maximilian Kang, Hyun-Seo Fallmann, Jörg Maticzka, Daniel Backofen, Rolf Stadler, Peter F. Zarnack, Kathi Sattler, Michael Legewie, Stefan König, Julian Genome Res Research 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. Cold Spring Harbor Laboratory Press 2018-05 /pmc/articles/PMC5932610/ /pubmed/29643205 http://dx.doi.org/10.1101/gr.229757.117 Text en © 2018 Sutandy et al.; Published by Cold Spring Harbor Laboratory Press http://creativecommons.org/licenses/by-nc/4.0/ This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
spellingShingle Research
Sutandy, F.X. Reymond
Ebersberger, Stefanie
Huang, Lu
Busch, Anke
Bach, Maximilian
Kang, Hyun-Seo
Fallmann, Jörg
Maticzka, Daniel
Backofen, Rolf
Stadler, Peter F.
Zarnack, Kathi
Sattler, Michael
Legewie, Stefan
König, Julian
In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title_full In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title_fullStr In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title_full_unstemmed In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title_short In vitro iCLIP-based modeling uncovers how the splicing factor U2AF2 relies on regulation by cofactors
title_sort in vitro iclip-based modeling uncovers how the splicing factor u2af2 relies on regulation by cofactors
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5932610/
https://www.ncbi.nlm.nih.gov/pubmed/29643205
http://dx.doi.org/10.1101/gr.229757.117
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