Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing. P re-mRNA splicing involves the removal of introns and ligation of the flanking exons. This reaction is catalyzed by the spliceosome, a macromolecular machine composed of five RNAs and hundreds of proteins (1). Alternative splicing generates multiple mRNAs from a single gene, thus increasing proteome diversity (2). Alternative splicing also plays a key role in the regulation of gene expression in many developmental processes ranging from sex determination to apoptosis (3), and defects in alternative splicing have been linked to many human disorders (4). In general, alternative splicing is regulated by proteins that associate with the pre-mRNA and function to either enhance or repress the ability of the spliceosome to recognize the splice site(s) flanking the regulated exon (5). Whether a particular alternative exon will be included or excluded from an mRNA in each cell is thought to be determined by the relative concentration of a number of positive and negative splicing regulators and the interactions of these factors with the pre-mRNA and components of the spliceosome (5).Although at least 74% of human genes encode alternatively spliced mRNAs (6), relatively few splicing regulators have been identified. Much of our insight into the mechanisms of splicing regulation was initially obtained by genetic analysis of the sex determination pathway in Drosophila (3). These experiments have identified three proteins, Sex-lethal (SXL), Transformer (TRA), and Transformer 2 (TRA2), that tightly regulate the alternative splicing of five genes, Sex-lethal, transformer, male specific lethal-2, fruitless, and doublesex. Subsequent biochemical experiments helped to elucidate the mechanisms by which SXL, TRA, and TRA2 function in this pathway. Aside from these examples, a simple genetic system to analyze specific alternative splicing events has not been available. Here we describe an RNA interference (RNAi) screen in cultured Drosophila cells designed to identify RNA-binding proteins that regulate alternative splicing of pre-mRNAs transcribed from endogenous ge...

Alternative splicing is generally controlled by proteins that bind directly to regulatory sequence elements and either activate or repress splicing of adjacent splice sites in a target pre-mRNA. Here, we have combined RNAi and mRNA-seq to identify exons that are regulated by Pasilla (PS), the Drosophila melanogaster ortholog of mammalian NOVA1 and NOVA2. We identified 405 splicing events in 323 genes that are significantly affected upon depletion of ps, many of which were annotated as being constitutively spliced. The sequence regions upstream and within PS-repressed exons and downstream from PS-activated exons are enriched for YCAY repeats, and these are consistent with the location of these motifs near NOVA-regulated exons in mammals. Thus, the RNA regulatory map of PS and NOVA1/2 is highly conserved between insects and mammals despite the fact that the target gene orthologs regulated by PS and NOVA1/2 are almost entirely nonoverlapping. This observation suggests that the regulatory codes of individual RNA binding proteins may be nearly immutable, yet the regulatory modules controlled by these proteins are highly evolvable.[Supplemental material is available for this article. The RNA-sequence data from this study have been submitted to the NCBI Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under accession nos. GSM461176-GSM461181.]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;. 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, 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 Barash et al. 2010;Zhang et al. 2010), a decipherable sequence-based information system that dictates ...

Alternative splicing is a powerful means of regulating gene expression and enhancing protein diversity. In fact, the majority of metazoan genes encode pre-mRNAs that are alternatively spliced to produce anywhere from two to tens of thousands of mRNA isoforms. Thus, an important part of determining the complete proteome of an organism is developing a catalog of all mRNA isoforms. Alternatively spliced exons are typically identified by aligning EST clusters to reference mRNAs or genomic DNA. However, this approach is not useful for genomes that lack robust EST coverage, and tools that enable accurate prediction of alternatively spliced exons would be extraordinarily useful. Here, we use comparative genomics to identify, and experimentally verify, potential alternative exons based solely on their high degree of conservation between Drosophila melanogaster and D. pseudoobscura. At least 40% of the exons that fit our prediction criteria are in fact alternatively spliced. Thus, comparative genomics can be used to accurately predict certain classes of alternative exons without relying on EST data.

Background: Histone methylation plays important roles in development and embryonic stem cell (ESC) differentiation. Results: Inhibition of the H3K9 demethylase JMJD1C directly down-regulated miR-302 and promoted neural differentiation of human ESCs (hESCs). Conclusion: JMJD1C inhibits neural differentiation of hESCs at least partially by epigenetically sustaining miR-302 expression. Significance: We provide novel evidence for epigenetic regulation of miR-302 to control neural differentiation of hESCs.

RNA interference (RNAi) is becoming a popular method for analyzing gene function in a variety of biological processes. We have used RNAi in cultured Drosophila cells to identify trans-acting factors that regulate the alternative splicing of endogenously transcribed pre-mRNAs. We have generated a dsRNA library comprising ~70% of the Drosophila genes encoding RNA binding proteins and assessed the function of each protein in the regulation of alternative splicing. This approach not only identiWes trans-acting factors regulating specific alternative splicing events, but also can provide insight into the alternative splicing regulatory networks of Drosophila. Here, we describe this RNAi approach to identify alternative splicing regulatory proteins in detail.