HNRNPA1 promotes recognition of splice site decoys by U2AF2 in vivo

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 noncoding regions (introns). One of the earliest events in this process is recognition of the 3′ splice site (3′ss) 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 proofreading 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 overexpression cells. We observed changes in the distribution of U2AF2 crosslinking sites relative to the 3′ss of alternative cassette exons but not constitutive exons upon HNRNPA1 overexpression. 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 noncanonical U2AF2 binding sites, Alu-derived RNA sequences represented one of the most abundant classes of HNRNPA1-dependent decoys. We propose that one way HNRNPA1 regulates exon definition is to modulate the interaction of U2AF2 with decoy or bona fide 3′ss.


Introduction
Precursor messenger RNA (pre-mRNA) splicing is catalyzed by a large macromolecular 48 complex composed of five uridine-rich small nuclear ribonucleoprotein particles (U snRNPs) and

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In this study, we investigate how HNRNPA1 influences the association of U2AF2 with

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Splicing reporter assays demonstrated that U2AF2 decoys sites are required for HNRNPA1-92 dependent splicing repression in HEK cells. Taken together our data suggest the intriguing 93 hypothesis that HNRNPA1 regulates alternative splicing by altering competition between bona 94 fide 3'ss and decoy sites for U2AF2 binding. Additionally, we discovered a role for mobile 95 U2AF2 decoy sites in splicing regulation, suggesting that Alu elements can play a major role in 96 the evolution of novel alternative splicing events.

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To determine how HNRNPA1 influences the association of U2AF2 with 3' splice sites on 101 a global scale, we established an HNRNPA1-inducible expression system in HEK293 cells. We 102 then assayed U2AF2 and HNRNPA1 protein-RNA interactions in control or HNRNPA1 over-

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1B and supplemental Fig 2A and B). In all cases, the immunoprecipitated material was both UV-   117   Table 4), we observed differences in the distribution of U2AF2 peaks across genomic locations 118 between control and HNRNPA1 over-expression cell lines (Fig. 1C). Most notably, the 119 proportion of peaks located in coding exons (CDS) or in exon-proximal intronic regions was 120 reduced whereas intronic peaks located more than 500 nt from exons (distal intron) increased.

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To determine whether HNRNPA1 over-expression influences the RNA binding specificity 124 of U2AF2, we searched for over-represented RNA sequences within the binding site peaks (Fig.   125   1D). In both control cells and HNRNPA1 over-expression cells, U2AF2 peaks are characterized 126 by a pyrimidine-rich motif, closely resembling authentic 3' splice sites. Although the sequences 127 are distinct from each other, SRSF1 and HNRNPA1 motifs also appear to be similar between 128 control and HNRNPA1 over-expression cells (Supplemental Figs. 2A Fig. 3), there are no differences in HNRNPA1 crosslinking sites between control 137 and over-expression cells (Supplemental Fig 4A and B, left panels, blue and red lines 138 respectively). SRSF1 crosslinking to exonic sequences was modestly reduced in the HNRNPA1 139 over-expression cells compared to control, but the positional distribution of the SRSF1 sites 140 relative to the 3'ss was largely unchanged for constitutive and skipped exons (Supplemental Fig   141   4A and B right panels, blue and red lines, respectively). U2AF2 crosslinking distribution relative 142 to the 3'ss was substantially altered in HNRNPA1 over-expressing cells compared to the 143 control, where a characteristic peak is observed over the 3'ss of both constitutive and skipped 144 exons ( Fig. 2A Tables 5 and 6). In regions with 150 no detectable HNRNPA1 crosslinks, the change in U2AF2 crosslinking exhibits a bimodal 151 distribution, which corresponds to regions flanking the 3'ss that show either increased or 152 decreased U2AF2 crosslinking in HNRNPA1 over-expression cells relative to control cells ( Fig.   153 2C, blue). By contrast, U2AF2 crosslinking to the vicinity of the 3'ss is significantly reduced 154 when a direct association of HNRNPA1 is also evident (Fig. 2C, pink). For example, in both 155 SRSF6 (serine/arginine-rich splicing factor 6 or SRp55) and PIEZO1 (Piezo-type 156 mechanosensitive ion channel component 1) we found that U2AF2 crosslinking near the 3' 157 splice sites is reduced in the cell lines over-expressing HNRNPA1 (Supplemental Figure 5).

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To determine if changes in U2AF2 crosslinking correlated with HNRNPA1-dependent 160 splicing regulation, we sequenced poly-A+ selected RNA libraries from control and HNRNPA1          manually curated the list of 83 splicing events that also had detectable U2AF2 crosslinking. We found that 41% of HNRNPA1-dependent exon skipping events exhibited redistribution of U2AF2 225 to adjacent Alu elements (Table 1). Taken together, these data demonstrate that over-226 expression of HNRNPA1 influences the association of U2AF2 with antisense Alu elements, 227 which may contribute to splicing regulation.

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We did not observe any change in hnRNP C protein expression or localization (Supplemental Fig.   291 1). Taken together, our data demonstrate that Alu-derived sequences function as RNA regulatory 292 elements that respond to changes to the intracellular concentration of splicing factors. It is 293 intriguing to speculate that, as primate-specific elements, antisense Alu elements may influence 294 the evolution of splicing regulation by modulating recognition of bona fide exons. Our results

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suggest the intriguing hypothesis that antisense Alu elements contribute to species-specific 296 differences in alternative splicing throughout the primate lineage.

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Strand-specific MEME-ChIP and HOMER analyses were performed on these sequences to find

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The authors declare that they have no competing interests.