Conservation of an RNA Regulatory Map Between Drosophila and Mammals

Alternative splicing is a process by which multiple messenger RNAs (mRNAs) can be generated by joining exons together in different combinations. This process is used to both increase protein diversity and to regulate gene expression (Nilsen and Graveley 2010). Approximately 95% of human genes contain introns and therefore have the potential to be alternatively spliced. Recent deep sequencing surveys of 10 human tissues found that nearly all (95%–98%) multi-exon human genes are alternatively spliced (Pan et al. 2008; Wang et al. 2008). Given the ubiquity of alternative splicing and the key roles it plays in the control of gene expression, it is important to develop a complete understanding of the mechanisms by which alternative splicing is regulated. Alternative splicing is most commonly controlled by RNA binding proteins that bind to sequence elements called enhancers and silencers (Nilsen and Graveley 2010). Splicing regulators bound to these enhancers or silencers are thought to either recruit or inhibit assembly or activity of spliceosomal components at nearby splice sites. The best-characterized splicing regulator proteins are the SR and hnRNP protein families. SR proteins primarily bind to enhancer sequences in exons where they activate adjacent splice sites, while hnRNPs have mostly been shown to suppress splicing when bound to intronic silencers. In addition to SR and hnRNPs proteins, several other splicing regulators have been identified that function in a tissue specific manner (Chen and Manley 2009). The mammalian proteins NOVA1 and NOVA2 (collectively named here as NOVA) are perhaps the best-characterized splicing regulators to date. NOVA1/2 encode RNA binding proteins with three KH-domains that recognize clusters of YCAY repeats. Over the past decade, several hundred splicing events have been shown to be regulated by NOVA1/2 (Ule et al. 2005, 2006; Licatalosi et al. 2008). A comparison of the locations of the NOVA1/2 binding sites with NOVA-regulated splicing events has revealed a stereotypical “RNA map” for NOVA1/2. Specifically, regions upstream of exons where NOVA inhibits splicing and regions downstream from exons where NOVA activates splicing were enriched with NOVA binding sites (Ule et al. 2006; Licatalosi et al. 2008). Similar “RNA maps” that link the position of binding sites to typical activities of the regulatory proteins have also been developed for mammalian FOX1/2 (Zhang et al. 2008; Yeo et al. 2009), PTB (Xue et al. 2009), and four D. melanogaster hnRNP proteins (Blanchette et al. 2009). Such maps, splicing expression data, and RNA sequence motifs have recently been used to predict regulated tissue-specific splicing changes in mouse, strongly supporting the existence of a splicing code (Wang and Burge 2008; Barash et al. 2010; Zhang et al. 2010), a decipherable sequence-based information system that dictates the splicing pattern of a given pre-mRNA under a specific condition. Though considerable progress has been made, interpreting this code remains a formidable task in the field. In particular, it is unclear how the mouse splicing code can be applied to different species, especially distantly related organisms such as Drosophila. Moreover, the extent to which the RNA maps of individual splicing regulators are static or plastic throughout evolution has been unknown. We were interested in exploring the conservation of the splicing code between distantly related organisms. As a first step in this process, we sought to generate an RNA map of Pasilla (PS) (Seshaiah et al. 2001), the D. melanogaster ortholog of NOVA1/2. To identify PS-regulated exons, we used RNA-seq (Wold and Myers 2008) to identify splicing events that changed upon depletion of PS by RNAi. We conclude that the RNA map of PS and NOVA1/2 is highly conserved between mammals and insects.