In all organisms, tRNAs are matured by a series of post-transcriptional processing events before they can partake in protein synthesis. These include end trimming to generate the correct 5' and 3' ends, and a substantial number of post-transcriptional chemical modifications; these ensure proper structure and function. Despite much effort many modifications have not been recapitulated in vitro, which have made understanding their mechanism difficult, if not impossible. One such event entails the C to U editing of tRNAs at the anticodon, a discovery that dates back to almost 25 years, but one for which no in vitro assay was available. Recently, we established the first in vitro assay for C to U editing in any system. Our studies also revealed that before editing can take place, the edited position must also be methylated and both enzymes, the deaminase and the methylase, strictly require each other for activity. This allowed us to introduce a new concept of ?enzyme co-activation?, one that may guide biochemical studies of other modifications for which no in vitro assay currently exist. Additionally, given that tRNA editing and modification in T. brucei is peppered with unique features, successful completion of these studies will generate important basic information on the mechanism and evolution of tRNA editing and modification, reveal unique features specific to T. brucei but which undoubtedly impact other systems. !

Public Health Relevance

Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo RNA editing and modification; pathways that are essential for viability. Although, editing and modification are not unique to trypanosome tRNAs, we have uncovered a remarkable case of two enzymes that converge at a single site and require each other for activity. This project will unveil the mechanism of enzyme co-activation in these medically important organisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM084065-09
Application #
9595466
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Reddy, Michael K
Project Start
2008-08-01
Project End
2022-05-31
Budget Start
2018-08-20
Budget End
2019-05-31
Support Year
9
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Ohio State University
Department
Microbiology/Immun/Virology
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
McKenney, Katherine M; Rubio, Mary Anne T; Alfonzo, Juan D (2018) Binding synergy as an essential step for tRNA editing and modification enzyme codependence in Trypanosoma brucei. RNA 24:56-66
Rubio, Mary Anne T; Gaston, Kirk W; McKenney, Katherine M et al. (2017) Editing and methylation at a single site by functionally interdependent activities. Nature 542:494-497
Kang, Byeong-Il; Miyauchi, Kenjyo; Matuszewski, Michal et al. (2017) Identification of 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNAs. Nucleic Acids Res 45:2124-2136
Fleming, Ian M C; Paris, Zden?k; Gaston, Kirk W et al. (2016) A tRNA methyltransferase paralog is important for ribosome stability and cell division in Trypanosoma brucei. Sci Rep 6:21438
McKenney, Katherine M; Alfonzo, Juan D (2016) From Prebiotics to Probiotics: The Evolution and Functions of tRNA Modifications. Life (Basel) 6:
Alfonzo, Juan D (2016) Post-transcriptional RNA modification methods. Methods 107:1-2
Lopes, Raphael R S; Silveira, Gilbert de O; Eitler, Roberta et al. (2016) The essential function of the Trypanosoma brucei Trl1 homolog in procyclic cells is maturation of the intron-containing tRNATyr. RNA 22:1190-9
Hauenschild, Ralf; Tserovski, Lyudmil; Schmid, Katharina et al. (2015) The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Res 43:9950-64
Lopes, Raphael R S; Kessler, Alan C; Polycarpo, Carla et al. (2015) Cutting, dicing, healing and sealing: the molecular surgery of tRNA. Wiley Interdiscip Rev RNA 6:337-49
Sample, Paul J; Gaston, Kirk W; Alfonzo, Juan D et al. (2015) RoboOligo: software for mass spectrometry data to support manual and de novo sequencing of post-transcriptionally modified ribonucleic acids. Nucleic Acids Res 43:e64

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