Abstract RNA editing in trypanosomes is a remarkable post-transcriptional process that results in the formation of mitochondrial mRNAs differing from their genes by the insertion or deletion of uridylyl (U) nucleosides. The information for RNA editing is provided by small guide RNAs (gRNAs) that basepair with their cognate pre-mRNAs to direct the precise sites for U-insertion or U-deletion. While considerable progress has been made in the elucidation of the components of the editing machinery and the general mechanism of RNA editing little is known about the regulation and function of this process. Simplistically, it has been thought that the sole function for RNA editing was to correct mistakes in mitochondrial protein coding genes thus allowing the translation of edited mRNAs to produce components of the mitochondrial respiratory system. While this is certainly an important function for RNA editing we recently discovered that primary mRNAs are differentially edited and translated to produce novel mitochondrial proteins. Our initial studies focused on the mRNA for cytochrome c oxidase III (COIII), which we discovered was alternatively edited in Trypanosoma brucei. A gRNA for the alternatively edited COIII transcript was identified and antibodies against the predicted novel coding sequence of the alternatively edited protein (AEP-1) reacted with a mitochondrial membrane protein. We have recently expanded the analysis of alternative mRNA editing to four additional genes, NADH dehydrogenase subunits 7, 8, 9 (ND7, 8, 9) and ATP synthase subunit 6 (A6). Alternatively edited mRNAs, creating novel open reading frames, were found for each of these genes. The overall goals of this proposal are to determine the extent of alternative mRNA editing in trypanosomes and the function mitochondrial proteins encoded by these RNAs. To accomplish these goals the following specific aims are proposed. In Specific Aim 1, we will examine the function of AEP-1 using a combination of biochemical and genetic approaches. In these studies, we will use dominant- negative mutants to evaluate the role of AEP-1 in kinetoplast DNA maintenance, proteomic analysis to identify associated proteins and in vitro DNA binding studies to further explore the function of this protein. Specific Aim 2, we will carryout a comprehensive gRNA and mRNA sequence analysis to examine the extent of potential mitochondrial protein diversity generated by alternative mRNA editing. Using conventional cDNA cloning and sequencing of mitochondrial mRNAs and 454 sequencing of gRNAs we will evaluate the extent of alternative RNA editing for mRNAs encoded by 12 mitochondrial genes. These data will be used to generate in silico RNA editing simulations to predict the magnitude of alternative mRNA editing. Putative alternatively edited mRNAs will be validated based on coding sequence, RNA stability and the identification of predicted protein products by proteomic, cell fractionation and reverse genetics. Together these studies will provide the first analysis of the role of RNA editing in protein diversification in trypanosomes and is likely to lead to the discovery of novel mitochondrial proteins.