The overall goal of this proposal is to study the biochemical processes involved in the regulation of embryonic muscle growth, particularly during the transition of embryonic to adult stages. Although myogenesis is primarily regulated at the transcriptional level, translational controls involving a variety of mRNAs in very early stages of embryonic development, are known to operate in a diverse number of systems. Our previous and ongoing work on the isolation, characterization and biological activity of a cytoplasmic translation inhibitory 10S ribonucleoprotein (iRNP) containing a 4S heterogeneous RNA (iRNAA) in the 65-10 nucleotide size range from chick embryonic muscle suggests that iRNA mediated translational inhibition may control cellular mRNA translation patterns in embryonic muscle. As shown by us and others, developmentally regulated isoforms of troponin T TnT), a component of the Ca2+- regulatory troponin (Tn) complex and generated by alternative splicing both at the 5' and 3' ends of a single TnT gene, are involved in the altered Ca2+ sensitivity, a physiologically relevant parameter of muscle during development. We have also identified highly conserved domains in all members of the TnT multigene family and shown by mutagenesis studies, their potential role in the organization and function of the Tn complex. The objectives of this project are too characterize the chicken TnT gene with respect to its evolution and organization of the exons that are constitutively and alternatively spliced and to correlate these studies with the expression of the protein isoforms; to delineate by using mutagenesis, the role of specific domains (e.g., conserved, """"""""embryo- specific,"""""""" etc.) in the function of TnT using in vitro assay systems for intersubunit interaction and Ca2+ sensitivity in reconstituted thin filament; to continue our work on the structure, function and mode of action of iRNA subspecies in order to correlate their potential role in regulating the expression of protein isoforms during muscle development. The proposed studies will increase our understanding of the biochemical correlates involved in changes of physiological properties of embryonic and adult skeletal muscles.
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