Scope and mechanism of coordinated alternative splicing and alternative polyadenylation

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Project Summary: In metazoan animals there is an incredible diversity of alternative transcript isoforms generated from genes. The alternative parts making up a mature full length mRNA include alternatively selected transcription start sites, 5′ UTRs, protein-coding exons, 3′ UTRs, and polyA sites. We have gained incredible insights into the cellular conditions and protein factors that regulate these alternative choices, but these regulatory events are usually studied in isolation. What is far less understood is how these alternative choices are coordinated together. This is partly because of limitations in the molecular techniques to study full length transcript isoforms. Long- read RNA sequencing technologies present a new tool to study how the alternative parts of a transcript come together. The majority of genes in metazoans undergo alternative polyadenylation to produce alternative length 3′ UTR isoforms. Long 3′ UTR isoforms are enriched in neural tissues, whereas short 3′ UTR isoforms are enriched in proliferating cells and in testis. In Drosophila, we recently identified that long 3′ UTR biogenesis and alternative splicing of the Dscam1 gene are co-regulated by the RNA-binding protein Elav in neurons. We found that these events are required for neuronal function in Drosophila. Our preliminary analysis shows that this coupling of alternative splicing changes to alternative 3′ UTR selection affects other genes in Drosophila. How widespread is the coupling of alternative exon selection to alternative 3′ end formation? Due to limitations in short-read RNA sequencing technologies, the genome-wide scope of such coupling events is unknown. We will employ new long read RNA-sequencing technologies to uncover 3′ UTR-coupled alternative splicing events genome-wide that are regulated by RNA-binding proteins such as Elav. What is the mechanism that coordinates 3′ UTR biogenesis with alternative splicing? In addition to co- regulation of alternative splicing and alternative polyadenylation of Dscam1 by Elav, we found that the presence of the Dscam1 long 3′ UTR was necessary for Elav-regulated alternative splicing. What is the nature of this “at a distance” regulatory event? A possible mechanistic explanation involves looping of the pre-mRNA such that the long 3′ UTR interacts with upstream splice sites to deliver RNA-binding proteins. We will test this pre-mRNA looping model through mapping RNA-RNA interactions, in vivo mini-gene reporter analyses, targeted screens, and RNA-affinity chromatography. This mechanistic investigation will be expanded to other genes and cell types. The proposed research program will identify the rules governing coordinated alternative splicing and alternative polyadenylation. The coordination of RNA processing steps to produce mRNAs with specific protein- coding and regulatory properties is likely important for many cell types and eukaryotic organisms, including humans.