, 2010; López de Silanes et al., 2004). These results reveal the utility of in vivo HITS-CLIP as a means of clarifying in vitro studies

of RNA-protein interactions, which here initially led to the skewed perception that nElavl proteins Ibrutinib supplier bind only to ARE elements (Table S6). We find that nElavl proteins in fact bind GU-rich elements relative to ARE elements by ∼1.3-fold and that it does so in clusters, analogous to the way in which Nova proteins recognize specific targets by binding clusters of low complexity YCAY elements (Licatalosi et al., 2008; Zhang et al., 2010). Previous studies in Drosophila have indicated that nElavl proteins are able to regulate alternative splicing ( Koushika et al., 2000; Lisbin et al., 2001; Soller and White, 2003, 2005). Prior studies of mammalian nElavl splicing regulation has been less clear, as neither comparisons in genetically modified animals nor direct RNA binding assays have been previously employed. Here, we combined nElavl-RNA direct binding data with bioinformatics and exon junction array data comparing splicing in WT and KO animals to identify a definitive set

of brain transcripts directly regulated by nElavl proteins in vivo. The results demonstrate that nElavl proteins directly bind neuronal pre-mRNA Epigenetic inhibitor to regulate alternative splicing and that the proteins have redundant actions in this regard, as splicing changes were uniformly more pronounced in DKO than Elavl3 or Elavl4 single KO brain. Our nElavl-RNA map is reminiscent of the position-dependence of splicing regulation observed for Nova, Fox2, hnRNP C, hnRNPL, TIA1/2, TDP-43, Mbnl, Ptbp1, and Ptbp2 and generally conforms to the finding that preferential binding to downstream introns leads to exon no inclusion, and to upstream introns exon exclusion (Licatalosi

et al., 2008, 2012; Llorian et al., 2010; Tollervey et al., 2011; Ule and Darnell, 2006; Yeo et al., 2009; Zhang et al., 2008). nElavl-mediated exon exclusion may be more frequently associated with binding to both upstream and downstream introns, a characteristic also noted for TDP-43 associated alternative splicing. As was also seen in the TDP-43 associated alternative splicing RNA-map, nElavl binding was observed in deeper intronic sequences of a small number of cassette exons. Our nElavl-RNA map is also in agreement with several candidate target gene studies examining the role of nElavl proteins in AS. For example, it was recently demonstrated that Elavl3 promotes inclusion of the alternatively spliced exon 6 of the Elavl4 gene by binding to U-rich sequences located in the intron downstream to the alternative exon ( Wang et al., 2010a).