REGULATED SPLICING OF MESSENGER RNA PRECURSORS

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The main objectives of this proposal are to identify and characterize the molecular factors and events that regulate alternative splicing of pre- mRNA in mammalian systems. The importance of understanding alternative splicing stems from its widespread role in specifying the fidelity and diversity of polypeptide synthesis and because this process is an important control point for gene expression at the post-transcriptional level. The specific aims that are the focus of the proposed research have been designed to address the mechanisms that specify alternative exon selection and alternative intron retention in model systems that are biologically relevant and amenable to experimental analysis. One avenue of research will address the question of how alternative exon selection is positively regulated by a mechanism that is proposed to involve template-directed, exon-bridging interactions between the essential splicing factors U1 snRNP and U2AF65. This work is a continuation of our previous studies of alternative splicing using the rat preprotachykinin gene as a model system. An important goal of this research is to reconstitute the exon-bridging complex with purified components and to use this reconstituted system to characterize the molecular interactions that are important for function (exon selection). A second avenue of research will explore the roles of essential splicing factors in the contrasting mechanism of alternative intron retention, which involves an unusual mini intron embedded in the messenger RNA encoding the transcriptional activator FosB. Splicing of the mini intron leads to the expression of a truncated protein with altered functional properties. A detailed analysis of cis-acting signals and trans-acting components will be carried out in vivo and in vitro to test the hypothesis that this mechanism involves a deactivation, or repression of splice site selection. An important goal of this research is to gain insight into the diverse roles of splicing factors that operate to select an intron in comparison to the mechanism of exon selection. The third avenue of research will address the question of cell-specific regulation of exon selection using the mammalian GABA A receptor gamma2 subunit gene. In this example, alternative splicing regulates the selection of a 24 nucleotide exon in various brain tissues. Alternative splicing of the gamma2 subunit gives rise to two protein isoforms that differ in the presence or absence of a phosphorylation site that is believed to play a pivotal role in the regulation of GABA A receptor function by protein kinases. The proposed research will dissect the sequence elements required for cell-specific exon selection and use this information to develop specific assays to screen for regulatory molecules. An important emphasis and long term goal of the proposed work is the isolation and characterization of cell-specific regulatory factors that direct alternative exon selection in the mammalian central nervous system.