Abstract We will elucidate how three RNA editing Catalytic Complexes (CCs) interact with Substrate Complexes (SCs), Accessory Factors (AFs), mRNAs and gRNAs to generate translatable mitochondrial mRNAs via precise U insertion and deletion and do so differentially between bloodstream (BF) and procyclic (PF) forms in Trypanosoma brucei. We hypothesize that this occurs via specific molecular interactions between domains of CC, SC and AF proteins and the mRNA/gRNA substrate. We will in: Aim 1. Identify these interactions in PFs and determine which are RNA dependent by a combination of in vivo proximity labelling, protein-protein cross- linking, and affinity purification methods. We will build structural models to identify similarities and differences between the three CCs. Aim 2. Determine the effects of loss of function (LOF) mutations of editing steps on these interactions, interactions with RNA by cross-linking immunoprecipitation and on RNA editing by RT-PCR sequencing. We will prioritize mutations that affect initiation of editing, subsequent catalytic steps and the functions of other essential proteins that are in all three CCs. This will validate and extend results from Aim 1, link the interactions to functional steps of editing and advance the structural models. Aim 3. Identify physical and functional interactions in BF cells by approaches used in Aims 1 and 2 and extended using a MGA knock-in approach which eliminates dependence on RNA editing in BFs and enables additional analyses of LOF mutations and interactions with RNA. This aim will identify similar and different molecular interactions between BFs and PFs. Aim 4. Determine the detailed characteristics of protein domains that function differently between BFs and PFs. We will determine the detailed chemical, structural, and functional characteristics of CC protein domains that affect editing differently in BFs vs. PFs using a focused saturation mutagenesis approach. Overall, this project will advance the understanding of how multiprotein complexes generate translatable mRNAs for essential components of the mt energy generating system in protozoan pathogens and do so differently between their developmental stages. The results may aid identification of targets for novel drug targets that are needed and provide insights into mechanisms of protein/nucleic acid interactions that are widespread and important in eukaryotes and which may lead to practical applications.