Coordination of alternative exon and 3'UTR choices in mRNAs

University Of Connecticut Sch Of Med/Dnt, Farmington CT

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Abstract

PROJECT SUMMARY/ABSTRACT With the advent of long-read RNA sequencing technology, there is now the opportunity to quantify how disparate alternative mRNA termini choices and internal exon choices are combined in full-length mRNAs. Recent evidence indicates coordinated interactions between alternative termini and exon choices in various organisms and developmental contexts, rather than random combinations. In Drosophila, individual genes have been found to exhibit tight coordination of both alternative first exons and alternative internal exons with alternative 3’UTRs. This project will investigate the role of cell type in coordinating these RNA processing events. A novel long read sequencing approach will be developed (scPL-Seq) that will permit the quantification and mechanistic interrogation of coordinated RNA processing events at the single-cell level during Drosophila neurodevelopment. The regulatory factors that coordinate these RNA processing events in specific cell types will be identified. In Drosophila, we previously demonstrated for the Dscam1 gene that coordination of alternative exon and 3’UTR choice is required for axon growth and neurodevelopment. In human neurons, the functional roles of specific exon-3’UTR combinations are unknown. The scope, regulation, and function of such coordinated events will be investigated in human ESCs differentiated to neurons. Alternative exon to 3’UTR choices will be quantified using a targeted adaptation of direct RNA sequencing (dRNA-target-Seq) that will provide not only connectivity of exonic content in full length mRNAs, but also isoform specific polyA tail lengths and RNA modification information. The factors regulating these coordinated events will be investigated, with an emphasis on neuronal RNA binding proteins. The functional roles of coordinated RNA processing events will be interrogated for specific genes, with an initial focus on transcriptional regulation genes. Preliminary work suggests that exon to 3’UTR coordination occurs for many human genes, including the de novo DNA methyltransferase DNMT3A. The importance of such RNA processing events and their coordination will be investigated in ES derived neurons using loss of function approaches. These pursuits will lead to accessible methods to study regulation of gene expression at the level of full-length mRNAs in diverse systems and single cells. Studying the function of alternative exon to 3’UTR coordinated events in human ES derived neurons will enable future investigations of how disease associated sequence variants affect mRNA processing in the context of full-length transcripts.