Intellectual Merit. RNA editing, the specific alteration of an RNA molecule relative to the gene from which it was copied, is widespread, and has been most extensively studied in mammals, plants, flies, viruses, and trypanosomes. 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 each of these forms of editing is the primitive eukaryote Physarum polycephalum (Pp). The sequence of virtually all of the mitochondrially-encoded mRNAs, rRNAs, and tRNAs in Pp differs from that of the mitochondrial genome, and a number of the 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. This project has two broad aims: the identification of factors that participate in nucleotide insertion and the adaptation of a newly developed mitochondrial transformation system for use in editing studies. The coupling of editing with RNA synthesis by the mitochondrial RNA polymerase inform the approaches to be used to enrich for editing factors, which include affinity selection of active transcription complexes and immuno-affinity purification of the polymerase and associated proteins. 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 advance the RNA editing field.
Broader Impacts: In addition to providing insight into RNA editing mechanisms, this project will provide training opportunities at many levels. The principal investigator is a former eighth grade science teacher committed to broadening the participation of under-represented groups in the sciences. She has significant experience mentoring high school women, minority students, and undergraduate students and will continue to participate in the highly successful Scientific Enrichment and Opportunity program for minority high school students and outreach programs geared towards minority undergraduates. The project will engage undergraduate and graduate students, as well as a postdoctoral associate, in various aspects of the research. Undergraduate students will carry out research throughout the academic year, culminating in a capstone research project during their senior year. Graduate students and the postdoctoral associate will learn optimization methods and master state-of-the-art techniques. Graduate students and post-docs will also be encouraged to participate in outreach activities and, when appropriate, participate in mentoring undergraduate students to gain relevant supervisory experience. Opportunities for developing presentation skills will be provided for all laboratory members through participation in lab meetings, student symposia, and regional and international meetings, with active encouragement and coaching by the principle investigator. Research results will be written jointly for dissemination to the larger scientific community.
