Molecular Cell
Volume 45, Issue 3, 10 February 2012, Pages 314-329
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Article
hnRNP A1 Proofreads 3′ Splice Site Recognition by U2AF

https://doi.org/10.1016/j.molcel.2011.11.033Get rights and content
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Summary

One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3′ end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3′ splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.

Highlights

► hnRNP A1 displaces U2AF from uridine-rich RNAs not followed by a 3′ splice site AG ► A 3′ splice site AG allows formation of a ternary complex with hnRNP A1 and U2AF ► AG proofreading requires U2AF35 and the glycine-rich domain of hnRNP A1 ► hnRNP A1-mediated proofreading influences U2 snRNP recruitment

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Present address: NMR Spectroscopy Research Group, Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands