Identification of alternative splicing regulators by RNA interference in Drosophila

Abstract

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...

A regulator of Dscam mutually exclusive splicing fidelity

“…Most notably, hrp36 associated with the entire exon 4, 6 and 17 clusters, but not the exon 9 cluster. This is consistent with the idea that hrp36 has a role in regulating exon choice in the exon 4 and 17 clusters 20 and mutually exclusive splicing of the exon 6 cluster while having no impact on the splicing of the exon 9 cluster. Thus, these results, in particular the association of hrp36 throughout the entire exon 6 cluster, are consistent with the observed effects of hrp36 on Dscam splicing.…”

“…For the RNAi screen we used a library of ~400 double-stranded RNAs corresponding to genes encoding RNA-binding proteins, spliceosomal components and DEXH/D-box ATPases 20 . The microarray screen identified seven proteins as candidates for controlling the fidelity of mutually exclusive splicing, as they modulated the splicing of most if not all exons within at least one of the clusters (Supplementary Discussion and Supplementary Fig.…”

“…2). Previously, we have shown that hrp36 has a role in alternative splicing of the exon 4 and 17 clusters of Dscam, but in these cases hrp36 controls which of the variable exons are selected rather than the number of exons included 20 . Consistent with these earlier results, when we used constitutive exon primers, we found no evidence that hrp36 RNAi results in the inclusion of multiple variable exons in the exon 4, 9 or 17 clusters ( Fig.…”

“…Transcription templates were generated by PCR from a plasmid library containing complementary DNA fragments from ~400 genes encoding mRNA processing factors 20 . Double-stranded RNA was synthesized by in vitro transcription (Epicentre T7 flash kit) and annealed by incubating at 100 °C for 2 min and slowly cooling the reactions to room temperature (20-22 °C) over a 40-min time period.…”

Use of RNA interference to dissect the roles of trans-acting factors in alternative pre-mRNA splicing

“…The RNAi screen we have conducted, which consisted of 250, or roughly 70%, of the Drosophila RNA binding proteins, led to the identification of 47 proteins that are involved in controlling the alternative splicing of 19 alternative exons from three different pre-mRNAs [9]. This type of screen can quickly lead to the identification of candidate splicing regulators of any pre-mRNA of interest.…”