Messenger RNA splicing is an essential step in eukaryotic gene expression and requires the action of the small nuclear ribonucleoprotein particles (snRNPs). These particles consist of a small nuclear RNA molecule (snRNA) which forms a complex with a set of specific proteins. Although the U1-U6 snRNPs are known to be essential for splicing, the specific functions of these snRNAs and their associated proteins are poorly understood. Our long term objective is to define the roles of the RNA and protein components of the snRNPs, and to discover how these molecules contribute to the consummate specificity of the splicing reaction. The goal of this proposal is to identify the precise function of the highly conserved 70K protein, a component of the U1 snRNP. During splicing, the U1 snRNP identifies 5' splice sites by forming complex interactions with the intron and with other snRNPs. It is not known how the RNA and protein components of the U1 snRNP contribute to this reaction. Yeast cells contain a 70K analog which is 33% identical to mammalian 70K. The proposed experiments will use the powerful genetic and biochemical techniques available in Saccharomyces cerevisiae to determine the function of the 70K protein. The function of 70K in splicing will be identified using a two-stage strategy. In the first stage, a set of conditional 70K mutants will be tested with general in vivo and in vitro assays to determine which aspects of splicing are affected by loss of 70K function. The second stage will then systematically test for 70K participation in detail using assays specific for different parts of the splicing reaction. These include 5' splice site identification, 5' cleavage site determination, branch site recognition, interaction with the U2 snRNP, and direct interaction with the pre-messenger RNA as well as with other RNAs and proteins involved in splicing. Analysis of 70K in yeast will give new information about RNA binding proteins and their roles in cellular processes. Due to the high conservation of 70K, information gained in yeast is expected to be directly applicable to mRNA splicing and RNA-protein interactions in higher systems. While the roles of the snRNAs in splicing have been investigated, to date, no functions have been identified for any snRNP protein. Therefore, discovery of the function of 70K will be a significant advance in our understanding of splicing.
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