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 region and the RNA binding domains 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 regions of the canonical RNA binding domains.
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 biophysical methods to identify a regulatory mechanism for U2AF2 RNA recognition. We found that the intrinsically disordered linker region connecting the two RNA recognition motif (RRM) domains 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 important role of intrinsically disordered linkers to modulate RNA interactions of multidomain RBPs.