Alternative splicing is an important mechanism for genetic control in eucaryotic cells. Through this process, a variety of proteins can be created from a single gene by altering the splicing pattern of the gene's primary transcript. Alternative splicing is especially prominent in the differentiation of the nervous system, where it regulates the production of a number of proteins that are important form neuronal development, function, and disease. Unfortunately, although our understanding of the general biochemistry of splicing has advanced significantly, little is known in any system, 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 a new exon (the N1 exon) into the src mRNA. In mapping the sequence determinants that regulate N1 exon splicing, a particularly interesting set of sequences, in the intron downstream of N1, was found to be required for the neuron-specific activation of the exon. The downstream of N1, was found to be required for the neuron-specific activation of the exon. The regulated splicing of src was reconstituted in vitro using extracts of neuronal and non-neuronal cells. In this in vitro splicing system, the neuron-specific activation of the N1 exon required proteins that bound to the downstream activator sequence. This project will pursue the molecular analysis of src neuron-specific splicing. In transfection experiments, the cis-acting elements that activate N1 splicing will be identified and characterized, through site specific mutagenesis. The mechanism of the repression of the exon in non-neuronal cells and its neuronal activation will be analyzed, at the level of spliceosome assembly, using the in vitro splicing system. The trans-acting factors that regulate src splicing will be identified by both their binding activity to specific RNA elements and by their activity in in vitro splicing assays. In the longer term, the purification of these proteins will lead to their cloning and study of how these splicing regulatory proteins affect neuronal differentiation. Our goal is to understand in molecular detail how the process of tissue- specific splicing is regulated. We will ultimately use this understanding of src neuron-specific splicing to look at its relation to other neuron-specific splicing events and at how this process of splicing control affects the program of neuronal differentiation.
