Alternative pre-mRNA splicing plays a major role in expanding the transcript output of human genes. This process is regulated, in part, by the interplay of trans-acting RNA binding proteins (RBPs) with myriad cis-regulatory elements scattered throughout pre-mRNAs. These molecular recognition events are critical for defining the protein coding sequences (exons) within pre-mRNAs and directing spliceosome assembly on non-coding regions (introns). One of the earliest events in this process is recognition of the 3′ splice site by U2 small nuclear RNA auxiliary factor 2 (U2AF2). Splicing regulators, such as the heterogeneous nuclear ribonucleoprotein A1 (HNRNPA1), influence spliceosome assembly both in vitro and in vivo, but their mechanisms of action remain poorly described on a global scale. HNRNPA1 also promotes proof reading of 3′ ss sequences though a direct interaction with the U2AF heterodimer. To determine how HNRNPA1 regulates U2AF-RNA interactions in vivo, we analyzed U2AF2 RNA binding specificity using individual-nucleotide resolution crosslinking immunoprecipitation (iCLIP) in control- and HNRNPA1 over-expression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3′ splice sites of alternative cassette exons but not constitutive exons upon HNRNPA1 over-expression. A subset of these events shows a concomitant increase of U2AF2 crosslinking at distal intronic regions, suggesting a shift of U2AF2 to "decoy" binding sites. Of the many non-canonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. Splicing reporter assays demonstrated that mutation of U2AF2 decoy sites inhibited HNRNPA1-dependent exon skipping in vivo. We propose that HNRNPA1 regulates exon definition by modulating the interaction of U2AF2 with decoy or bona fide 3′ splice sites.

HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo