Recent studies indicate that alternative splicing plays a major role in regulating gene expression during heart development, however the extent of regulation and the physiological roles for developmental splicing transitions are unknown. Alternative splicing is regulated by RNA binding proteins that bind within the pre- mRNA near the regulated splice site(s) to enhance or repress splicing. This project focuses on the CUGBP, Elav-like family (CELF) of RNA binding proteins that is down-regulated ten-fold during postnatal heart development promoting expression of a subset of protein isoform transitions due to alternative splicing. CELF- regulated targets are enriched for genes that function in endocytosis and vesicular trafficking that we hypothesize reflects a role in postnatal maturation of the cellulr components for excitation contraction coupling (ECC). We will determine the functions of protein isoforms expressed during postnatal cardiomyocyte development using cell culture and existing CELF gain of function lines of mice. We are using postnatal development to identify isoform transitions that have a physiological impact since a high fraction are conserved and like transcriptional programs, splicing programs revert to fetal patterns in heart disease. We will firs use antisense oligonucleotides to redirect splicing of vesicular trafficking genes in two cell line including a large scale screen of redirected splicing to identify protein isoform transitions with physiological impact. We will then use systemic delivery of antisense oligonucleotides targeting a limited set of high priority genes for reversion to fetal splicing patterns in the heart. Of particular interest will be genes that revert to fetal splicing patterns in heart failure. Our goalis to establish correlations between isoform transitions and structure and function of the ECC apparatus. Given the lack of understanding of the normal roles of developmental splicing programs, the knowledge gained from this project will increase our understanding of both normal development and pathogenic mechanisms in congenital and adult heart disease.
Alternative splicing plays a larger role in controlling gene expression than previously appreciated. This proposal studies the physiological consequences of alternative splicing during postnatal heart development. The results will provide new information regarding normal heart function that is critical to strategically develop therapeutic approaches to reverse or circumvent disease mechanisms.
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