Pre-mRNA splicing is an essential step in the expression of most mammalian genes. A number of human genetic diseases have been shown to be the result of mutations affecting pre-mRNA splicing. The long-term objective of the research proposed here is to understand the mechanisms of mammalian pre-mRNA splicing and its regulation. The approach is to study the function of a group of metazoan splicing factors containing arginine and serine rich sequences (the RS domain) which play important roles in both constitutive and alternative splicing. Previous work has demonstrated specific interactions among this group of proteins, suggesting mechanisms for the 5' and 3' splice sites association through protein-protein interactions and for alternative splicing factors to function by directly interacting with components of the constitutive splicing machinery. This proposal aims to characterize the protein-protein interaction domains of the RS domain containing splicing factors, to test the functional significance of protein-protein interactions in pre-mRNA splicing, to identify new splicing factors, to determine the roles of these proteins in constitutive and regulated alternative splicing. Preliminary studies have revealed existence of two protein-protein interaction domains in a single RS domain containing protein. Functional significance of the protei-protein interactions observed was suggested by the inhibition of pre-mRNA splicing by a dominant negative form of this protein. The feasibility of identifying new splicing factors was demonstrated by studies of three putative splicing factors, one identified by its structural characteristics and two isolated by their interactions with a known splicing factor. Experiments are also proposed to investigate the possible roles of the RS domain containing proteins in regulating the alternative splicing of Ich-1 pre-mRNA. Ich-1 is a gene involved in programmed cell death; and alternative splicing of Ich-1 pre-mRNA gives rise to functionally antagonistic products. An assay for Ich-1 alternative splicing in cultured cells will be established; and differential roles of splicing factors on Ich-1 splicing will be studied. The proposed studies should provide significant new insights into the roles of mammalian splicing factors in constitutive and regulated alternative splicing, and may also advance our understanding of programmed cell death.
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