Alternative splicing is an important means of genetic control in eucaryotic cells. By altering the splicing pattern of the primary transcript, a variety of proteins can be created from a single gene. Alternative splicing is especially prominent in the mammalian nervous system, where it regulates the production of many proteins that are important for neuronal development, function, and disease. Unfortunately, although our understanding of the general biochemistry of splicing has advanced significantly, much less is known of how splicing is regulated. The mouse c-src gene has provided an effective model system for studying a neuron-specific splicing event. Neurons produce a different form of the src protein from other tissues, resulting from the neuron-specific insertion of an extra exon (the N1 exon) into the src mRNA. Two major cis-acting elements that control N1 splicing have been identified by site specific mutagenesis of a transfected src mini-gene. These are an intronic splicing enhancer, downstream of N1, that activates splicing of the exon but is only partially neural specific, and the 3' splice site upstream of the exon that represses the splicing in non-neuronal cells. The combination of these two sequences confers neural specific splicing on a heterologous test exon. The regulated splicing of the N1 exon was reconstituted in extracts of neuronal and non-neuronal cells and some of the regulatory proteins have been identified. These include the KSRP, hnRNP F, hnRNP H and PTB proteins binding to the downstream enhancer, and the PTB protein also binding to the upstream 3' splice site. How the RNA/protein complexes at these sites combine to generate the precise tissue specific inclusion of an exon is still unclear. This project will pursue the molecular analysis of src neuron-specific splicing. Using a variety of biochemical assays, the assembly of the regulatory proteins onto the enhancer and repressor RNA sequences will be studied, and the interactions of these RNA/protein complexes with each other, and with components of the spliceosome will be dissected. New regulatory proteins will be characterized, including a neural specific form of PTB. The role of the recently described splicing enhancer protein, KSRP, will be studied and the functional domains of the protein delineated. Finally, simplified splicing systems will be developed for the analysis of individual regulatory proteins. Our goal is to understand in molecular detail how a simple change in splicing pattern is regulated in differentiated cells.
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