The goal of this research is to identify factors responsible for RNA editing. RNA editing involves specific alterations of RNA molecules, relative to the genes from which they were copied, and affects expression of genes in mitochondria, the organelles responsible for energy generation in the cell. Whereas, RNA editing is widespread in many organisms, including humans, the underlying mechanisms are not well understood. This research will fill that knowledge gap and provide new insights how RNA editing occurs. The project will also provide training opportunities at many levels through concerted efforts to involve high school, undergraduate, and graduate students. One such effort will be to continue ongoing work with a high school teacher and students to develop experiments that can be done in an inner city school setting. Specific outreach to women and minority students will help broaden participation in science. Students will take part directly in the research, gaining valuable experience by learning how to optimize methods, master state-of-the-art techniques, and present the results of their work as part of the project's dissemination of the research outcomes to the broader scientific community.
RNA editing occurs in many organisms and has been most extensively studied in humans and other mammals, plants, and human pathogens. Observed sequence changes include base conversions, base substitutions, the deletion of encoded nucleotides, and the insertion of additional residues. The only organism known to carry out all of these forms of editing is the primitive eukaryote Physarum polycephalum. The sequence of virtually all of the mRNAs, rRNAs, and tRNAs encoded in Physarum mitochondria differs from that of the mitochondrial genome in highly specific and reproducible ways. Many RNAs contain so many non-encoded nucleotides that their genes escaped identification for years. Previous work has shown that "extra" nucleotides are added to these RNAs during their synthesis and that the specificity of editing depends on template elements that flank editing sites; however, the underlying mechanism is still mysterious. The major aim of this project is to identify the factors that are required for these site-specific changes to mitochondrial RNAs. Previous work on two different forms of editing has narrowed the list of potential candidates for each type and these will be investigated further. The expression of each of these proteins will be knocked down individually and mitochondrial RNAs will be analyzed to determine whether RNA editing levels are reduced. Once editing factors are identified, their interactions with other mitochondrial proteins will be examined. Long-term goals for the project include the development of an in vitro system assembled from defined components for use in mechanistic studies and the definition of the specific roles played by both template elements and auxiliary factors. Successful completion of these studies will significantly enhance current understanding of how RNA sequence changes can be targeted in an exquisitely specific manner.
