RNA editing is a process involving highly specific, localized changes within RNA molecules, leading to sequence differences between the RNA and the gene from which it is transcribed. Editing occurs in a broad range of biological systems and, along with alternative splicing, provides a means of generating multiple protein products from a limited set of genes. Many editing events are regulated, and mistakes in editing have been linked to human diseases and disorders. The long-term goal of this project is the elucidation of the mechanism of insertional RNA editing in Physarum mitochondria. The major aims of this proposal are the identification of the cis-acting elements and the trans-acting factors required for accurate insertion of non-templated nucleotides into Physarum mitochondrial RNAs. The role of potential cis-acting elements will be examined using an established in vitro system that allows the creation of chimeric templates that contain defined changes. Based on previous work, a subset of the proteins that interact with the mitochondrial genome and the mitochondrial RNA polymerase are likely to be involved in editing. Trans-acting factors associated with the mitochondrial genome, as well as proteins that interact with the polymerase, will be isolated using genetic, biochemical, and affinity strategies. A range of functional and immunological assays will be used to determine which of these factors are likely to play a role in Physarum RNA editing. Because insertional editing in Physarum mitochondria occurs as the RNA is being made, these experiments should lead to a better understanding of both this novel mode of RNA synthesis and maturation and the means by which transcription elongation can be used to modulate gene expression.
