The objective of this proposal is to determine the molecular basis for the regulation of pre-mRNA alternative splicing. Alternative splicing plays a crucial role in differentiation and development, and expression of specific alternatively spliced variants is characteristic of many human diseases. Despite the importance of alternative splicing to normal cellular function, little is known about the mechanisms that direct tissue-specific regulation. This proposal continues our investigation of the cardiac troponin T (cTNT) pre-mRNA in which inclusion of a cassette alternative exon predominates in embryonic striated muscle and skipping predominates in the adult. Exon inclusion in embryonic muscle requires conserved intronic elements located upstream and downstream of the exon. Multiple copies of individual elements promote muscle-specific inclusion of a heterologous exon in embryonic striated muscle. The mechanism of regulation is complex involving antagonist activities of positive and negative-acting factors which bind adjacent sites within these elements. A role for PTB as a repressor of cTNT exon inclusion in muscle and nonmuscle cells has been established. The positive-acting factors include members of a novel family of RNA-binding proteins expressed in different tissue-specific patterns. These proteins activate element-dependent exon inclusion when coexpressed with cTNT minigenes in nonmuscle cells. Element-dependent inclusion has been reconstituted in a cell-free splicing assay using recombinant proteins. Changes in the expression of one of these proteins correlates with changes in cTNT splicing during skeletal muscle differentiation and heart development. The main goals of this proposal are to: (i) characterize the regulatory functions of the positive-acting factor expressed during striated muscle differentiation and development. (ii) determine the molecular mechanism of element- dependent exon inclusion using the established in vitro splicing assay. (iii) isolate and identify the components of the RNP complexes associated with exon skipping and exon inclusion. Genetic and biochemical approaches will be used to identify the critical interactions that link muscle-specific auxiliary splicing elements to the basal splicing machinery. Insights gained from these studies will be directly applicable to basic molecular mechanisms that affect human health.
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