The long-range goal of our work is to understand the molecular basis for alternative RNA splicing using the rat beta-tropomyosin (TM) gene as a model system. This gene expresses two different isoforms via alternative splicing, namely: (1) skeletal muscle beta-TM and (2) fibroblast TM-1, which is also expressed in smooth muscle where it corresponds to smooth muscle beta-TM. At present the molecular basis for this tissue-specific expression of the beta-TM gene is not known. Tissue-specific splicing will be studied in vivo by transfection of mini-genes and in vitro by splicing of SP6 polymerase derived pre-mRNAs, to determine which sequences in the pre-mRNA contain the necessary information for alternative RNA splicing. Using UV cross linking, gel mobility shift, and direct binding assays, RNA-binding proteins that interact with the critical cis-acting elements will be identified. In vitro complementation assays will be used to identify the factor(s) in nonmuscle cells that is required for the use of certain 3'-splice sites as well as factors that block the use of the skeletal muscle exon. Nuclear extracts derived from myogenic cells will be used to identify and study both cis-acting elements and cellular factors involved in muscle-specific RNA splicing. These studies will provide important information of how alternative splicing of the beta-TM gene is controlled in skeletal muscle and nonmuscle cells. The studies outlined in this proposal will have broad implications for numerous other biological systems that involve alternative RNA processing as a mechanism to regulate gene expression. Alternative RNA processing for the generation of protein diversity has been reported for a large number of cellular genes and DNA and RNA viruses. Understanding the various levels of TM gene expression will provide valuable clues to a more general understanding of how tissues such as skeletal muscle, the heart, and smooth muscle are developmentally regulated. Changes in gene expression are ultimately responsible for changes in the contractile properties of skeletal and cardiac muscles and other cell types observed in different developmental, hormonal, physiological and pathological states. In order to understand the molecular basis for these developmental and patho-physiological changes, it is essential to understand the regulation of gene expression at the molecular level. Since alternative RNA splicing is a fundamental process, it remains to be determined if any diseases or abnormalities are associated with defects in the cellular splicing machinery. Such information will be useful in terms of diagnostic and preventive intervention, and in the development of new therapeutic agents.
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