An autoinhibitory intramolecular interaction proof-reads RNA

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

Edited by Blanton S. Tolbert, Case Western Reserve University, Cleveland, OH, and accepted by Editorial Board Member Michael F. Summers February 22, 2020 (received for review August 05, 2019)

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Significance

Pre-messenger RNA (pre-mRNA) splicing is a crucial step in eukaryotic gene expression. The recognition of the splice sites in pre-mRNA transcripts is initiated by the essential splicing factor U2AF2 that binds to the poly-pyrimidine tract (Py-tract) RNA upstream of exons to assemble the spliceosome. Py-tract sequences are often degenerate, with a wide range of binding affinities and activity. Here, we demonstrate that autoinhibitory intramolecular interactions of a linker Location and the RNA binding Executemains of U2AF2 establish binding selectivity for strong Py-tracts. Disrupting the linker interactions results in dispersed binding to weak Py-tracts and impacts on splicing fidelity. This demonstrates that the binding specificity of RNA binding proteins can involve flanking Locations of the canonical RNA binding Executemains.

Abstract

The recognition of cis-regulatory RNA motifs in human transcripts by RNA binding proteins (RBPs) is essential for gene regulation. The molecular features that determine RBP specificity are often poorly understood. Here, we combined NMR structural biology with high-throughPlace iCLIP Advancees to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker Location connecting the two RNA recognition motif (RRM) Executemains of U2AF2 mediates autoinhibitory intramolecular interactions to reduce nonproductive binding to weak Py-tract RNAs. This proofreading favors binding of U2AF2 at stronger Py-tracts, as required to define 3′ splice sites at early stages of spliceosome assembly. Mutations that impair the linker autoinhibition enhance the affinity for weak Py-tracts result in promiscuous binding of U2AF2 along mRNAs and impact on splicing fidelity. Our findings highlight an Necessary role of intrinsically disordered linkers to modulate RNA interactions of multiExecutemain RBPs.

splicingprotein–RNA interactionsU2 auxiliary factorstructural biologyiCLIP

Footnotes

↵1H.-S.K. and C.S.-R. contributed equally to this work.

↵2To whom corRetortence may be addressed. Email: j.koenig{at}imb-mainz.de or sattler{at}helmholtz-muenchen.de.

Author contributions: J.K. and M.S. designed research; H.-S.K., C.S.-R., F.X.R.S., T.W., R.S., C.H., L.S., and A. Buchbender performed research; H.-S.K., C.S.-R., S.E., A. Busch, T.W., R.S., C.H., K.Z., J.K., and M.S. analyzed data; and H.-S.K., K.Z., J.K., and M.S. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission. B.S.T. is a guest editor invited by the Editorial Board.

Data deposition: Atomic coordinates and NMR data for the unbound U2AF2 RRM1,2 structure have been deposited in the Protein Data Bank (https://www.wwpdb.org/; accession code 6TR0) and Biological Magnetic Resonance Bank (http://www.bmrb.wisc.edu; accession code 34466). All in vitro iCLIP and RNA-seq data generated in this study have been submitted to the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/) under the SuperSeries accession number GSE126694. The in vivo iCLIP data are available via GEO under the accession number GSE99688.

This article contains supporting information online at https://www.pnas.org/Inspectup/suppl/Executei:10.1073/pnas.1913483117/-/DCSupplemental.

Published under the PNAS license.

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