This project will elucidate critical processes that precisely edit mt mRNAs in Trypanosoma brucei and its regulation and differential editing between life cycle stages. It examines the editosome endonuclease subcomplex which discriminates among thousands of editing sites (ESs), initiates editing and is a likely point of regulation. It uses novel cell lines and methods to examine three editosome endonucleases and their associated proteins and the roles of their domains, interactions and posttranslational protein modifications. It characterizes: 1. Editosome endonuclease ES specificity in vivo by determining the effects on editosome integrity, composition and function and on edited mRNAs of eliminating each endonuclease and mutating their RNase III, Zn finger and PUF domains in bloodstream (BF) and procyclic (PF) stages and in a BF line that survives without editing. It will quantify all editing in BFs and PFs, in vivo endonuclease specificity and if it differs between lif cycle stages. 2. The roles of endonuclease associated proteins by approaches similar to Aim 1. These include B4 and B5 and their RNase III, PUF, Zn finger domains to determine if their effect on endonuclease activity and mRNA cleavage;B6, B7 and B8 and their Zn fingers to determine their effects on endonuclease specificity and regulation;and the low abundance B9 and B10 that transiently interact with editosomes and may regulate their functions. These studies will determine the roles of these proteins in the specificity and regulation of ES selection and cleavage and between life cycle stages. 3. Physical and function interactions between endonucleases and associated proteins by complimentary in vivo and in vitro protein crosslinking methods. It will examine the roles of protein interactions in ES selection, steps of editing, editing of multiple sites and between life cycles. It will characterize the physical and functional organization of editosomes whether proteins exchange between editosomes during editing especially when editing of insertion and deletion ES specified by one gRNA. 4. The roles of editosome protein post-translational modification (PTM) by identifying sites of phosphorylation, methylation, ubiquitination and sumoylation. It will assess roles of PTM in editing and its regulation by examining the effects of mutating selected PTM sites on editosomes and editing and between life cycle stages. These studies will determine whether PTMs regulates steps in editing and editing between life cycle stages. Overall, the project will expand the understanding of a process that is essential for the parasite and may provide drug targets.

Public Health Relevance

This project will characterize critical features of multiprotein complexes that control the expression of essential genes in a group of lethal parasites. These complexes are not in humans and thus may be drug targets. However, characterization of their functional organization and molecular interactions will enhance understanding of how other RNA/protein complexes function, including many in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
3R01AI014102-37S1
Application #
8760040
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
1978-09-01
Project End
2017-12-31
Budget Start
2014-05-01
Budget End
2014-12-31
Support Year
37
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Seattle Biomedical Research Institute
Department
Type
DUNS #
City
Seattle
State
WA
Country
United States
Zip Code
98109
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 23:608-17
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
Carnes, Jason; Lerch, Melissa; Kurtz, Irina et al. (2015) Bloodstream form Trypanosoma brucei do not require mRPN1 for gRNA processing. RNA 21:28-35
Cestari, Igor; Stuart, Ken (2015) Inositol phosphate pathway controls transcription of telomeric expression sites in trypanosomes. Proc Natl Acad Sci U S A 112:E2803-12
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
Demir, Ozlem; Labaied, Mehdi; Merritt, Chris et al. (2014) Computer-aided discovery of Trypanosoma brucei RNA-editing terminal uridylyl transferase 2 inhibitors. Chem Biol Drug Des 84:131-9
Beck, Kirsten; Acestor, Nathalie; Schulfer, Anjelique et al. (2013) Trypanosoma brucei Tb927.2.6100 is an essential protein associated with kinetoplast DNA. Eukaryot Cell 12:970-8
Guo, Xuemin; Carnes, Jason; Ernst, Nancy Lewis et al. (2012) KREPB6, KREPB7, and KREPB8 are important for editing endonuclease function in Trypanosoma brucei. RNA 18:308-20

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