The long-term goal of this project is to understand in molecular terms how a simple change in mRNA splicing pattern is regulated in differentiated cells. Primary gene transcripts are often spliced in alternative patterns to produce a variety of mRNAs and proteins from a single gene. Alternative splicing is especially common in the mammalian nervous system where many proteins important for neuronal development and activity are expressed in multiple functional isoforms through changes in splicing. Several neurologic diseases, including Spinal Muscular Atrophy and a form of Frontotemporal Dementia, arise from errors in alternative splicing and the misproduction of particular spliced isoforms. In spite of its importance in cell differentiation, physiology, and disease, the mechanisms controlling splice site choice are poorly understood. The proposed work will continue our analysis of the positive and negative regulation of the c-src N1 exon in mammalian cells. We will relate our findings on this relatively simple example of alternative splicing to more complicated systems. We plan to characterize the structure and assembly of the Polypyrimidine Tract Binding Protein (PTB) complex that represses N1 exon splicing in non-neuronal cells. We will determine the role of the new neural PTB protein in altering this PTB complex and perhaps allowing the derepression of N1 splicing in neuronal cells. We will analyze the mechanism of splicing stimulation by the intronic splicing enhancer downstream of the N1 exon. Finally, we plan to characterize the large pre-mRNP complexes that contain a mini src pre-mRNA and that serve as the substrate for the splicing reaction in vivo and in vitro. From this work we hope to understand in precise molecular detail how a variety of combinatorial inputs can determine a change in splicing.
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