The long term objective of this project is to understand the mechanisms of nuclear pre-mRNA splicing, a fundamental process that takes plac in the nucleus of all eukaryotic cells and is a required step for the expression of most cellular and viral protein-coding genes. To this end, biochemical and molecular approaches will be employed for a detailed analysis of the structure and mechanism of action of selected human proteins required for splicing. These studies will focus on several RNA-binding proteins and on their role in recognizing and activating exonic elements that function as potent splicing enhancers. A specific protein phosphatase that appears to be required for splicing will be characterized. Biochemical methods will also be used to identify, isolate, and characterize novel human proteins required for splicing. Biochemical complementation will be sued to identify and purify activities required for the function of certain splicing enhancers. A similar approach will be used to identify essential factors in a recently discovered minor splicing pathway, through which a small class of introns with unique conserved elements is processed. Insights into the evolutionary and mechanisti relations between major and minor spliceosome pathways should be obtained. Errors in splicing fidelity resulting from mutations in intron-containing gene are the molecular basis of many genetic diseases. These mutations also affect splicing fidelity in vitro, and therefore, the mechanisms responsible for the remarkable specificity of splicing are amenable to biochemical analysis. Genetic defects in the expression or structure of cellular splicing factors might be associated with inherited diseases and cancer. Inefficient splicing i an essential feature of the life cycle of retroviruses, including HIV. Antisense modified oligonucleotides complementary to cryptic splice sites can correct aberrant splicing associated with thalassemia mutations in human beta-globin genes. Suppressor small nuclear RNAs have been engineered to rescu defective splice sites upon transfection into cultured mammalian cells. The spliceosome, a multienzyme complex, and its individual constituents should be explored as potential targets for novel therapeutic agents.
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