Sequence alterations that change the meaning of a tRNA in decoding are part of a growing number of post-transcriptional changes collectively known as tRNA editing. Editing can be influenced by the structural context of an editing site and in the case of tRNA can be modulated by posttranscriptional modifications. In trypanosomatids, tRNAs are transcribed in the nucleus, exported to the cytoplasm, and later a subset of cytoplasmic tRNAs is actively imported into the mitochondria. However, before tRNAs can be rendered functional in any cellular compartment, they face many enzymatic reactions including end trimming, intron splicing, tRNA editing, and chemical modification. Some of these processes, for example those involved in trimming of extraneous sequences at the tRNA ends, occur in the nucleus, usually preceding cytoplasmic export. Others, like editing and modification, may occur at any point in the tRNA maturation pathway and in any of the tRNA-containing compartments. Despite much progress made in last few years, it is still not clear how editing and modification pathways are integrated both at the molecular and cellular levels. We have proposed that tRNA editing and modification events can be highly coupled and exploited by some organisms to control gene expression at the level of tRNA specificity. This is especially important in single-cell eukaryotes like trypanosomatid parasites where is well accepted that the bulk of the genetic regulation occurs post-transcriptionally. In this proposal, we have continued our studies on the very unique tRNA editing enzyme of T. brucei but now prompted by our newly solved crystal structure we ask new questions of what makes this enzyme so unique. We also focus on a newly discovered set of methyltransferases, which surprisingly target the editing site providing a wonderful testing ground for our hypothesis of the interrelation of editing and modification and what this coupling may mean in terms of cellular function. Significantly, reconstitution of methylation activity in vitro requires addition of the recombinant editing enzyme a finding that is without precedent in the tRNA editing and modification field. As essential steps in tRNA maturation in trypanosomatids (Leishmania and Trypanosoma), these types of editing and modification events also provide very attractive targets for therapeutic intervention against parasites of very major medical importance. Given the link between tRNA maturation and disease, these studies will further expand our knowledge of the role tRNA takes as a central player in cellular metabolism. !

Public Health Relevance

Members of the genus Leishmania and Trypanosoma infect millions of people worldwide. In these organisms, tRNAs undergo post-transcriptional modifications and editing changes that are unique to this system. The enzyme responsible for tRNA editing changes in trypanosomatids possesses substrate specificities that are not shared with any other member of this family of proteins. We have now discovered two new enzymes (tRNA methylases) whose functions are tightly coupled to the previously described editing deaminases. The propose studies will determine the basis for substrate specificity of the T. brucei enzymes which in the future may open doors towards the development of drugs against this unique and essential activities. These studies will also provide functional and evolutionary insights into important members of the cytidine deaminase (CDA) superfamily.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1-IDM-S (02))
Program Officer
Bender, Michael T
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Ohio State University
Schools of Arts and Sciences
United States
Zip Code
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

Showing the most recent 10 out of 30 publications