The removal of intervening sequences from messenger RNA precursors (pre-mRNAs) by the process of splicing is an essential step in the expression of the vast majority of protein-coding genes in eukaryotic organisms. Errors in splicing have been demonstrated to be the molecular lesion in several human diseases, such as the beta-thalassemias. The many examples of tissue-specific and developmentally regulated alternative splicing are evidence of the central role pre-mRNA splicing plays in the control of eukaryotic gene expression. The long-term objective of the proposed research is to characterize the biochemical mechanism of pre-mRNA splicing in the fission yeast Schizosaccharomyces pombe. To make progress toward this goal, the specific aims for the next funding period are (1) to characterize the prp3+ gene; (2) to clone the prp6+ and prp7+ genes; (3) to select pre-mRNA splicing mutants that express an exon-skipping phenotype; (4) to characterize the protein components of the S. pombe spliceosomal snRNPs; (5) to investigate the interaction between the U2 and U6 snRNAs; and (6) to isolate endogenous spliceosomes. Two general experimental strategies are employed in the proposed research. First, genetic methods will be used to identify, clone and characterize genes required for pro-mRNA splicing. Second, immunochemical and direct biochemical analyses will be applied to investigate components of the spliceosome, with particular emphasis on the snRNPs. These small nuclear ribonucleoprotein particles form the core of the spliceosome and are recognized as antigens by patients suffering from the autoimmune disease lupus erythematosus and-other connective tissue disorders. The yeast system offers a combined genetic and biochemical approach that will facilitate the achievement of the immediate objectives and long term goals of the proposed work.
