REGULATED SPLICING OF MESSENGER RNA PRECURSORS

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The long term goal of this project is to understand the mechanism by which a tissue-specific splicing event is regulated. This requires elucidation of both cis-acting sequences and diffusible cellular factors involved. The approach taken to achieve these goals is to study the splicing of the preprotachykinin gene as a model for a tissue-specific splicing event. This is the simplest model available for these studies. The focus of this proposal is the apparently regulated splicing event that results in the skipping of the penultimate 3' exon preferentially in brain, in contrast to thyroid tissue. Specifically, this application proposes the following three aims. (1) A tissue culture model system will be developed to study preprotachykinin gene expression in cell lines that mimic the way in which this gene is differentially expressed in brain and thyroid tissues. (2) The sequence requirements for this exon-skipping event will be elucidated by directed mutagenesis of sequences in the region of the gene containing the optional exon. This will include deletion of intervening sequences and mutagenesis of splice site regions. The role of RNA stability and nuclear transport will also be addressed in studies using these cell lines to determine the extent to which this splicing event directs the production of the different cytoplasmic preprotachykinin mRNA forms. (3) The required cellular factors will be elucidated by biochemical studies using soluble cellular extracts and model pre-mRNA substrates constructed using the information gained from the mutational studies tested in the cultured cell lines. The foundation for understanding this tissue- specific splicing event will be provided by biochemical studies that test the intrinsic efficiency of individual splice sites within the context of splice sites derived from well characterized pre- mRNAs. The understanding of a regulated, tissue specific splicing event is critical for a complete understanding of developmental processes and tissue-specific gene regulation.