INTEGRATIVE GENOME-WIDE ANALYSIS OF ALTERNATIVE PRE-MRNA SPLICING REGULATION BY THE DROSOPHILA SR PROTEIN FAMILY

Abstract

Alternative pre-mRNA splicing is a powerful mechanism that is exploited by higher eukaryotes to diversify their proteomes, and to differentially regulate the expression, function, and localization of mRNA and proteins. Pre-mRNA splicing is typically regulated by RNA-binding proteins that recognize cis-acting RNA elements, and either activate or repress splicing of adjacent exons in a temporal, and tissue specific, manner. Understanding how RNA-binding proteins control the splicing code is fundamental to understanding organismal development and disease. The SR proteins are a well-conserved class of RNA-binding proteins that have an essential role in the regulation of splice site selection, and have also been implicated as key regulators during other stages of RNA metabolism. The complexity of the RNA targets, and specificity of RNA binding location remains poorly understood for many members of the SR protein family. Here, we present a comprehensive study to elucidate how the SR proteins coordinate to regulate alternative pre-mRNA splicing (AS) in Drosophila. Genome-wide analysis of SR-dependent splicing by RNA-seq, reveals that SR proteins are required for the regulation of many types of alternative splicing events, and can act as positive or negative regulators of splice site choice depending on their binding location on the target RNA. In addition, a vast majority of regulated targets require multiple SR protein members for regulation. RNAi of multiple SR proteins simultaneously results in an additive change in the magnitude of splicing. This indicates that SR proteins co-regulate alternative splicing events in a combinatorial manner through binding specific locations on the target transcripts. Using single-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP), we define the specific RNA-binding maps of the entire family of SR proteins in the transcriptome of Drosophila S2 cells. We find that SR proteins bind a distinct, but functionally diverse, class of RNAs that includes mRNAs, both constitutive and alternatively spliced, as well as non-coding RNAs. Closer analysis of the bound transcripts revealed that while individual SR proteins can bind unique transcripts, multiple SR protein family members bind a majority of the target transcripts. This comprehensive analysis reveals position-dependent RNA splicing maps, in vivo consensus binding motifs, and a high level of cross- and coordinated regulation of alternative splicing by the SR protein family.