Abstract

RNA editing is a novel and essential gene regulatory mechanism in trypanosomes that alters mitochondria! RNAs by specific uridine insertion and deletion to form functional mRNAs. The genetic information for editing is transferred from frans-acting gRNAs to mRNAs through base-pairing interactions. While considerable progress has been made in understanding the structure and composition of the core editing machinery, regulatory factors that affect the accuracy and efficiency of the editing process remain elusive. Our laboratory recently provided genetic evidence for the first RNA editing regulatory factori'the RNA binding protein, RBP16. RNAi-mediated knock-down of RBP16 in procyclic form (PF)trypanosomes leads to a dramatic and specific decrease in editing of apocytochrome b (CYb) RNA. CYb gRNA abundance is unaffected in RBP16 knock-downs, suggesting that regulation takes place at the level of gRNA utilization. Consistent with its role in vivo, RBP16 reproducibly stimulates RNA editing in vitro up to 5-fold. Preliminary data suggest that RBP16 enhances editing by an intriguing two-step mechanism, one aspect of which is independent of specific, high affinity RBP16-RNAinteractions. Our goal is to determine the scope and mechanism of RBP16 RNA editing regulation.
In Aim 1, we will down-regulate RBP16 expression in bloodstream form (BF)trypanosomes and analyze the effects on BF-specific and constitutive editing events. We will test the hypothesis that RBP16 facilitates recruitment of specific gRNAs to editosomes by analysis of editosome-associated gRNAs in PF and BF RBP16 knock-down cells.
In Aim 2, we will directly test the model that RBP16 stimulates both pre- and post-mRNA cleavage steps of editing. The role the RNP1 and RGG RNA binding domains of RBP16 in this process will be examined by comparison of mutant and wild type RBP16 in the in vitro assay.
In Aim 3, RBP16-interacting proteins will be identified by both expression of TAP-tagged RBP16 in PF and BF T. brucei and by a yeast two-hybrid screen. This combined genetic and biochemical approach will provide important insight into the largely unexplored area of RNA editing regulation.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI061580-04
Application #
7535268
Study Section
Pathogenic Eukaryotes Study Section (PTHE)
Program Officer
Mcgugan, Glen C
Project Start
2005-12-01
Project End
2010-11-30
Budget Start
2008-12-01
Budget End
2009-11-30
Support Year
4
Fiscal Year
2009
Total Cost
$294,315
Indirect Cost

  Institution

Name
State University of New York at Buffalo
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260

  Publications

Zimmer, Sara L; Simpson, Rachel M; Read, Laurie K (2018) High throughput sequencing revolution reveals conserved fundamentals of U-indel editing. Wiley Interdiscip Rev RNA :e1487
McAdams, Natalie M; Simpson, Rachel M; Chen, Runpu et al. (2018) MRB7260 is essential for productive protein-RNA interactions within the RNA editing substrate binding complex during trypanosome RNA editing. RNA 24:540-556
Kafková, Lucie; Debler, Erik W; Fisk, John C et al. (2017) The Major Protein Arginine Methyltransferase in Trypanosoma brucei Functions as an Enzyme-Prozyme Complex. J Biol Chem 292:2089-2100
Simpson, Rachel M; Bruno, Andrew E; Bard, Jonathan E et al. (2016) High-throughput sequencing of partially edited trypanosome mRNAs reveals barriers to editing progression and evidence for alternative editing. RNA 22:677-95
Read, Laurie K; Lukeš, Julius; Hashimi, Hassan (2016) Trypanosome RNA editing: the complexity of getting U in and taking U out. Wiley Interdiscip Rev RNA 7:33-51
Kumar, Vikas; Madina, Bhaskara R; Gulati, Shelly et al. (2016) REH2C Helicase and GRBC Subcomplexes May Base Pair through mRNA and Small Guide RNA in Kinetoplastid Editosomes. J Biol Chem 291:5753-64
Shaw, Porsha L R; McAdams, Natalie M; Hast, Michael A et al. (2015) Structures of the T.?brucei kRNA editing factor MRB1590 reveal unique RNA-binding pore motif contained within an ABC-ATPase fold. Nucleic Acids Res 43:7096-109
Huang, Zhenqiu; Faktorová, Drahomíra; K?ížová, Adéla et al. (2015) Integrity of the core mitochondrial RNA-binding complex 1 is vital for trypanosome RNA editing. RNA 21:2088-102
McAdams, Natalie M; Ammerman, Michelle L; Nanduri, Julee et al. (2015) An arginine-glycine-rich RNA binding protein impacts the abundance of specific mRNAs in the mitochondria of Trypanosoma brucei. Eukaryot Cell 14:149-57
Ammerman, Michelle L; Tomasello, Danielle L; Faktorová, Drahomíra et al. (2013) A core MRB1 complex component is indispensable for RNA editing in insect and human infective stages of Trypanosoma brucei. PLoS One 8:e78015

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