Pre-mRNA splicing is a basic and essential step in the transfer of information from gene to protein in mammalian cells, where nearly every gene transcript undergoes the removal of large amounts of non-essential information as it matures into a functional messenger RNA. As an indispensable step in gene expression, splicing presents a target for intervention to prevent the expression of unwanted genes (e.g., in cancer). In addition, the splicing process has gone away in many cases of genetic disease. A major part of this proposal is directed at a basic question regarding the mechanism of pre-mRNA splicing; how are splice sites recognized, how are the 100-base exons singled out from among 10,000 bases of introns in a primary transcript? Mammalian cell genetics will be used to perturb pre-mRNA structure to reveal changes that interfere with the splicing process. Toward this end, use will be made of dilhydrofolate reductase (dhfr) minigenes as selectable reporters that function only when splicing is disrupted, allowing the rapid screening of thousands of mutations to select those few changes that are effective. In another project, the effect of mutations in an endogenous adenine phosphoribosyltransferase (aprt) gene will be analyzed in great detail, documenting the steps in splicing that are blocked in different mutants and the effect that the order of intron removal may play in splice site selection. Splicing may occur in specialized regions of the nucleus; the effect of gene position on splicing efficiency will be tested in cells harboring a single copy of a transfected dhfr gene, each integrated into a different sites in the genome. The previous finding that premature termination of translation interferes with RNA processing will be studied by examining nuclear mRNA metabolism and transport of mRNA out of the nucleus in nonsense mutants. Alternative splicing is a process by which more than one functional protein can be produced from a single gene. Alternative splicing is a major determinant of cell differentiation and of viral growth. The mechanism by which a given gene transcript is spliced one way in one cell type and another way in another cell type will be studied by the molecular cloning of factors that inhibit one splicing pattern. In one system being studied, a gene that is expressed in the thyroid as the peptide hormone calcitonin, which regulates calcium metabolism, is expressed in neurons as the neuropeptide CGRP, which uses a different splicing pattern. A cloned cDNA will be isolated that codes for a factor produced in neurons that repress the calcitonin splicing pattern in favor of the CGRP pattern. This cDNA will be selected using mammalian cell genetic manipulations involving a selectable dhfr minigene. A similar approach will be used to isolate a cDNA for a factor that causes alternative splicing of alpha-tropomyosin transcripts in different types of muscle tissue. The cloned cDNA molecules will serve as a starting point for characterizing the mechanism of alternative splicing and the regulation of this process in development.
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