: This project will characterize the editing cycle in Trypanosoma brucei, the functions of editosome components that perform these steps, the regulation of editing during the life cycle, and the molecular and physiological consequences of editing in its inactivation. 1. The proposed studies will produce and characterize editosome integrity, composition and function in vivo and invitro from T. brucei in which genes for editosome components have been deleted, placed under regulated expression, mutated and/or affinity tagged. They will identify mutants that are blocked at specific steps of editing and provide insight into the functions of editosome genes, which will be directly tested. 2. Time course studies will characterize editosome changes in editosome composition and in vivo and in vitro function following inactivation and reactivation of normal and mutated genes that block editing prior to endonuclease cleavage. Editosome composition and function will also be characterized in mutants that do not edit in vivo due to the lack of gRNA and/or mRNA genes to assay for catalytic or structural RNA and assess the possible presence and state of editosomes. These studies are designed to determine the sequence of events that occur during the initiation of editing and assess whether it occurs by association of the RNAs with preformed editosomes, association of editosome subunits, or by de novo assembly of the RNAs and proteins. 3. Additional time course studies will characterize changes in editosome composition and in vivo and in vitro function following inactivation and reactivation of normal and mutated editosome genes that affect the catalytic and post-catalytic steps of editing. These studies will identify the genes with roles in catalysis and determine the sequence of events that occur during editing at a single site and during the decoding of single and multiple gRNAs. 4. The composition and in vivo and in vitro functional differences between normal and mutant editosomes from bloodstream and procyclic stages will be characterized to determine the process by which editing is differentially regulated during the life cycle. They will assess whether regulation occurs through changes in editosome composition, association with proteins, mRNA and/or gRNA, and/or due to stage-specific differences in editosome functionality. 5. Cellular RNA, protein, and metabolic changes that occur upon inactivation of editosome genes will be assayed to characterize the processes that lead to the death of BFs when editing is blocked. Similar experiments will compare wild type trypanosomes with viable mutants that do not exhibit editing due to kDNA deletions to uncover processes that allow their survival. We anticipate that these studies will uncover novel adaptive processes. This project will elucidate the process of editing, its regulation during the life cycle and assess its physiological significance, and hence the potential utility of editing as a drug target.
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