The overall goal of the proposed research is to study detailed aspects of general pre-mRNA splicing in mammalian cells, with an emphasis on the role of selected human protein splicing factors. We will continue the structural and functional characterization of SF2/ASF and related SR proteins, which are required for the early stages of splicing, including splice-site selection and spliceosome assembly. We will extend our studies of the interactions between SR proteins and exonic splicing enhancers, and investigate the structural basis for specificity, and the role of the various domains of the SR proteins. We will further investigate the function of a type-2C phosphatase as a splieeosome-assembly factor, and characterize its substrates and interacting proteins. In addition, we will complete the purification of, and functionally characterize, a human protein activity that appears to be conditionally required for splicing. Finally, we will study specific aspects of mRNA surveillance to further understand the relationship between stop codons and alterations in splicing or mRNA stability. Aberrant splicing due to mutations in intron-containing genes is often the cause of human genetic diseases. Because the same mutations usually also affect splicing specificity in vitro, the molecular basis of splicing specificity is amenable to biochemical analysis. Genetic defects in the expression or structure of cellular splicing factors may also be associated with inherited diseases and cancer. Inefficient splicing, another aspect of splicing specificity, is an important feature of the life cycle of retroviruses, including HIV. It may be possible to identify or design drugs that change the concentration or biochemical properties of specific spliceosome constituents in such a way as to compensate for decreased or aberrant splicing in mutant genes. It is already feasible to modulate the use of specific splice sites or exons through antisense methods, certain small molecules, or a new class of chimeric compounds. The proposed studies, which are aimed at understanding basic cellular mechanisms of gene expression, may provide the basis for the development of new diagnostic tools and therapeutic agents. ? ?
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