: 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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Tropical Medicine and Parasitology Study Section (TMP)
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Rogers, Martin J
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Seattle Biomedical Research Institute
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Cestari, Igor; Anupama, Atashi; Stuart, Kenneth (2018) Inositol polyphosphate multikinase regulation of Trypanosoma brucei life stage development. Mol Biol Cell 29:1137-1152
Cestari, Igor; Stuart, Ken (2018) Transcriptional Regulation of Telomeric Expression Sites and Antigenic Variation in Trypanosomes. Curr Genomics 19:119-132
Carnes, Jason; McDermott, Suzanne M; Stuart, Kenneth (2018) RNase III Domain of KREPB9 and KREPB10 Association with Editosomes in Trypanosoma brucei. mSphere 3:
Carnes, Jason; McDermott, Suzanne; Anupama, Atashi et al. (2017) In vivo cleavage specificity of Trypanosoma brucei editosome endonucleases. Nucleic Acids Res 45:4667-4686
McDermott, Suzanne M; Stuart, Kenneth (2017) The essential functions of KREPB4 are developmentally distinct and required for endonuclease association with editosomes. RNA 23:1672-1684
Cestari, Igor; Haas, Paige; Moretti, Nilmar Silvio et al. (2016) Chemogenetic Characterization of Inositol Phosphate Metabolic Pathway Reveals Druggable Enzymes for Targeting Kinetoplastid Parasites. Cell Chem Biol 23:608-617
McDermott, Suzanne M; Luo, Jie; Carnes, Jason et al. (2016) The Architecture of Trypanosoma brucei editosomes. Proc Natl Acad Sci U S A 113:E6476-E6485
McDermott, Suzanne M; Carnes, Jason; Stuart, Kenneth (2015) Identification by Random Mutagenesis of Functional Domains in KREPB5 That Differentially Affect RNA Editing between Life Cycle Stages of Trypanosoma brucei. Mol Cell Biol 35:3945-61
McDermott, Suzanne M; Guo, Xuemin; Carnes, Jason et al. (2015) Differential Editosome Protein Function between Life Cycle Stages of Trypanosoma brucei. J Biol Chem 290:24914-31
Carnes, Jason; Anupama, Atashi; Balmer, Oliver et al. (2015) Genome and phylogenetic analyses of Trypanosoma evansi reveal extensive similarity to T. brucei and multiple independent origins for dyskinetoplasty. PLoS Negl Trop Dis 9:e3404

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