Group II introns are mobile genetic elements that are the likely evolutionary ancestors of both the spliceosome and retrotransposons. RNA splicing and genomic change caused by retrotransposition are fundamental processes common to all eukaryotes. In this proposal, we will examine the mechanisms that drive the function of group II introns, and extend our findings back to their eukaryotic counterparts. Group II introns are comprised of a catalytic RNA core that binds to an intron-encoded protein (IEP) to form a ribonucleoprotein (RNP) complex. Splicing proceeds through two competing reactions: hydrolysis or branching. Introns with a minimal RNA architecture splice exclusively through hydrolysis; however, the addition of the IEP switches the splicing reaction to the branching pathway. This results in the formation of branched lariat RNPs capable of intron mobility. The overall goal of this application is to determine the detailed molecular mechanisms of how intron RNPs form and how these RNPs spread introns to new locations in DNA. This goal will be achieved by combining biochemical and structural biology approaches through the execution of the following two aims. 1) Determine the biochemical mechanism and structural basis for hydrolytic ribozyme activity and protein stimulated lariat-RNP formation. This will allow a detailed comparison of the active sites of a pure RNA self-splicing intron, self-splicing intron RNP and the spliceosomal RNP, providing the first insights into how the splicing machinery evolved from a pure ribozyme to the protein-RNA molecular machine responsible for processing almost all mRNA transcripts in the human transcriptome. 2) Determination of the biochemical mechanism and structural basis for intron mobility into DNA. These experiments will provide systematic mechanistic insights into the interplay between the intron and the IEP reverse transcriptase that complete the intron integration reaction into new DNA locations.
Although many human diseases are caused by mis-splicing of genes, we still do not fully understand the fundamental mechanisms that drive the function of the splicing machinery. Gene disruption caused by retroelement insertions is a significant source of gene mutation that causes human disease. Our proposed examination of group II intron splicing and retrotransposition will provide structural and mechanistic understanding of these fundamental processes, and lay the foundation for the development of RNA-based therapeutics.