The broad, long-term objectives of this research project are to study RNA metabolism and gene expression. Areas such as RNA processing , RNA transport, and RNA stability and degradation will eventually be explored. Initially, the process of pre-mRNA splicing (i.e., the precise excision of introns from messenger RNA precursors) will be studied. Specifically, the in vitro pre-mRNA splicing system that we developed in the yeast, Saccharomyces cerevisiae, will be utilized to identify and characterize the components in the splicing machinery (i.e., the extrinsic splicing factors and the functional spliceosomes). The results of this study should provide clue to health-related problems which are resulted from abnormal gene expression due to defects in the RNA splicing process. Three extrinsic protein factors required to convert the pre-mRNA on a functional spliceosome to spliced products have been identified by analyzing extracts isolated from several temperature-sensitive rna mutants. Efforts will be made to purify these extrinsic factors by biochemical fractionation of wild-type or overproducer extracts. Antibodies against purified proteins will be raised. The corresponding genes will be isolated by screening yeast DNA library with the antibodies or synthetic oligonucleotides. DNA sequences will be determined, the homology with other genes searched, and the derived protein sequences analyzed. The purified extrinsic factors will be used to examine and to define the functional spliceosomes isolated under different conditions. The roles of ATP in the splicing pathway will be investigated. Attempt will be made to identify splicing factors which may be phosphorylated or adenylated during the conversion of the spliceosome. The interactions among the spliceosome, the extrinsic factors, and the RNA will be investigated by using immunoprecipitation and RNase protection assays. Ultimately, the machinery for nuclear pre-mRNA splicing may be revealed.
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