The long term objective of this proposal is to understand the precise mechanism of nuclear pre-mRNA splicing, a fundamental process that takes place in all eukaryotic cells, and is a required step for the expression of most cellular and viral protein-coding genes. Our approach is to purify and characterize selected components of the human pre-mRNA splicing machinery, which consists of multiple small nuclear ribonucleoprotein particles (snRNPs) as well as multiple polypeptides. We are developing assays and reagents to facilitate the isolation of these components in their active forms, and to study how they interact with synthetic pre- mRNA substrates in a reconstituted in vitro splicing reaction. We have already purified the bulk U snRNPs by immunoaffinity chromatography with a monoclonal antitrimethylguanosine antibody we developed for this purpose. The snRNPs are recovered in active form, as determined by biochemical complementation. We have begun to fractionate some of the reburied protein components, which are also assayed by complementation for splicing. We propose to fractionate the snRNPs into individual active species, which will provide new information regarding the involvement of major and minor snRNPs, and the roles of their RNA and protein subunits in splicing. Selected protein components will also be purified further to study their participation in splicing together with the snRNPs. Purified components will be characterized structurally, and their mechanism of action will be studied in detail by biochemical methods. Errors in the specificity of splicing caused by mutations in intron-containing genes are often responsible for several human genetic diseases. The same mutations also affect the specificity of splicing in vitro. Therefore, the molecular basis for the specificity of splicing, which is currently unknown, is amenable to biochemical analysis. We will investigate the mechanisms of splice site selection by systematically analyzing sequence-specific interactions between purified splicing factors and wild type and mutant pre-mRNAs. In addition, we will investigate the possible existence of intron-specific splicing factors by studying the factor requirements for the splicing of wild type and thalassemic human beta-globin pre-, mRNAs, and of pre-mRNAs from two human viruses: adenovirus 2 and human immunodeficiency virus 1. Identification of such factors would have broad implications for our understanding of general splicing, and of developmentally and tissue-specifically regulated alternative splicing.
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