RNA splicing is a critical step in gene expression, involving excision of introns and covalent joining of exons from an RNA transcript. Specific RNA-RNA interactions are key features of splicing; they are essential for both splice site selection and catalysis of the splicing reactions. To date, studies on self-splicing group I introns have identified major structural features responsible for the catalytic activity of these ribozymes. However, two critically important and long-standing problems regarding splicing of group I introns remain unsolved. We do not know how 3' splice sites are selected; neither do we know how the catalytic core of the intron interacts with the splice sites. We have recently discovered a novel two base-pair interaction (termed P9.0) between the catalytic core of some group I introns and intron sequences at the 3' splice site, and have shown experimentally that this interaction exists and is functionally important in the exon ligation step of splicing (Burke, J.M. et al. Nature, in press). The discovery has allowed the P.I. to propose a tripartite model for 3' splice site selection (Burke, J.M. FEBS, Lett. 250, 129-133 (1989)) and puts us in a position to finally solve the 3' splice site problem. The specific objectives of this proposal are to (l) Determine the detailed structural basis for 3' splice site selection in group I introns, and (2) Use this information to develop a transacting ribozyme based on 3' splice site cleavage reactions. The project will provide insights on RNA splicing, catalysis and structure that will be of general importance to those studying splicing, biological catalysis by RNA, RNA structure, and the role of RNA structure in the mechanisms and regulation of gene expression. In addition, the ribozymes developed may have important applications as tools for in vitro manipulation of RNA, and as gene-specific agents.
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