The long term goal of this proposal is to understand the m o lecular basis for the regulation of pre-mRNA alternative splicing. Alternative splicing is a common and key regulatory step for the expression of diverse protein isoforms according to cell-specific or developmental programs. Despite significant advances in the understanding of the biochemistry of pre-mRNA splicing, little is known about the molecular components or mechanisms that regulate splice site selection. The PI will continue his investigation of developmentally regulated alternative splicing using the chicken cardiac troponin T (cTNT) gene in which a single alternative exon (exon 5) is included in embryonic striated muscle and skipped in the adult. During the last funding period we have made significant progress with regard to both constitutive and alternative splicing mechanisms. The investigator's accomplishments include: (I) identification and characterization of a previously unknown constitutive splicing element (referred to as a splicing enhancer) located within cTNT exon 5 and other alternative and constitutive exons; (ii) demonstration that a subset of the SR protein family of essential splicing factors binds to the enhancer and activates splicing; (iii) identification of an intronic element that is necessary and sufficient to activate muscle-specific exon inclusion in vivo; and (iv) reconstitution of a muscle-specific splicing in a cell free complementation system. A major focus of this proposal is to identify, isolate, and characterize factors that activate exon inclusion in embryonic striated muscle. A muscle-specific activator sequence has been localized within a 142 nucleotide segment immediately downstream of the alternative exon. The critical determinants within this region will be defined. A prime target is a conserved sequence found in two similarly regulated mammalian genes. The established in vitro complementation system will be used to characterize muscle-specific splicing activity. Functionally significant RNA-binding proteins will be identified using an approach that directly correlates in vitro RNA-binding with in vivo splicing activity. In vitro splicing and RNA-binding will be used as complementary assays to identify, characterize and, ultimately, isolate by cDNA cloning the factors that regulate a cell-specific splicing event. An understanding of cTNT alternative splicing could provide a paradigm for regulatory mechanisms in vertebrates. Regulation of alternative splicing is a fundamental process required for normal development and cellular function. Alterations of alternative splicing pathways are associated with pathologic changes in a number of diseases. Therefore, insights gained from these studies will be directly applicable to basic molecular mechanisms that affect human health.
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