The tRNAHis guanylyltransferase (Thg1) is absolutely essential in yeast, and likely throughout all eukaryotes, due to the universal requirement for G-1 on tRNAHis in all eukaryotes in which it has been investigated. Thg1 adds G-1 to tRNAHis via an unusual non-templated 3'-5'nucleotide addition reaction, by an unknown catalytic mechanism that cannot be predicted based on similarity to known enzymes, and thus is likely to employ a novel catalytic mechanism. Moreover, we have recently demonstrated that all Thg1 family members catalyze a template-dependent 3'-5'addition reaction with various substrates, and that this activity is used for a form of G-1 addition in archaea, as well as for an unusual tRNA editing reaction in protozoa. These demonstrated roles for templated 3'-5'addition greatly expand the scope of catalytic activities exhibited by Thg1 family members. Nonetheless, the presence of Thg1 homologs in archaea and bacteria that do not require enzymatic G-1 addition to tRNAHis and unexplained Thg1-related phenotypes in yeast and human cells suggest that additional roles for 3'-5'addition are yet to be uncovered. This application proposes the use of kinetic, genetic, biochemical and structural techniques to investigate the molecular mechanisms and biological functions of both non-templated and templated 3'-5'addition reactions catalyzed by diverse Thg1 family members. These results will provide insight into catalysis of a novel and apparently widespread, but largely unexplored, reaction in biology, and will enable further investigation into alternative functions for 3'-5'nucleotide addition in biological systems.

Public Health Relevance

Investigation of the unusual 3'-5'nucleotide addition reactions catalyzed by Thg1 family members is of importance to human health, due to the absolute biological requirement for Thg1 activity for tRNAHis function in eukaryotes including humans, the recently demonstrated link between Thg1 overexpression and diabetic nephropathy, and the possibility of discovering novel pathways of mitochondrial 5'-tRNA editing and/or repair, defects in which could contribute to the pathology of human diseases. Moreover, a detailed understanding of the mechanism of Thg1 catalysis in diverse organisms may lead to identification of unique properties of Thg1 homologs from significant human pathogens, such as Plasmodium falciparum and Trichomonas vaginalis, which can be targeted for the development of new antiparasitic or antifungal agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM087543-04
Application #
8516527
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Hagan, Ann A
Project Start
2010-08-15
Project End
2015-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
4
Fiscal Year
2013
Total Cost
$278,227
Indirect Cost
$91,934
Name
Ohio State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
832127323
City
Columbus
State
OH
Country
United States
Zip Code
43210
Long, Yicheng; Abad, Maria G; Olson, Erik D et al. (2016) Identification of distinct biological functions for four 3'-5' RNA polymerases. Nucleic Acids Res 44:8395-406
Edvardson, Simon; Elbaz-Alon, Yael; Jalas, Chaim et al. (2016) A mutation in the THG1L gene in a family with cerebellar ataxia and developmental delay. Neurogenetics 17:219-225
Rao, Bhalchandra S; Jackman, Jane E (2015) Life without post-transcriptional addition of G-1: two alternatives for tRNAHis identity in Eukarya. RNA 21:243-53
Jackman, Jane E (2015) An origin story: ribozyme catalysis by the ribosome. RNA 21:650-1
Long, Yicheng; Jackman, Jane E (2015) In vitro substrate specificities of 3'-5' polymerases correlate with biological outcomes of tRNA 5'-editing reactions. FEBS Lett 589:2124-30
Smith, Brian A; Jackman, Jane E (2014) Saccharomyces cerevisiae Thg1 uses 5'-pyrophosphate removal to control addition of nucleotides to tRNA(His.). Biochemistry 53:1380-91
Abad, Maria G; Long, Yicheng; Kinchen, R Dimitri et al. (2014) Mitochondrial tRNA 5'-editing in Dictyostelium discoideum and Polysphondylium pallidum. J Biol Chem 289:15155-65
Betat, Heike; Long, Yicheng; Jackman, Jane E et al. (2014) From end to end: tRNA editing at 5'- and 3'-terminal positions. Int J Mol Sci 15:23975-98
Jackman, Jane E; Alfonzo, Juan D (2013) Transfer RNA modifications: nature's combinatorial chemistry playground. Wiley Interdiscip Rev RNA 4:35-48
Hyde, Samantha J; Rao, Bhalchandra S; Eckenroth, Brian E et al. (2013) Structural studies of a bacterial tRNA(HIS) guanylyltransferase (Thg1)-like protein, with nucleotide in the activation and nucleotidyl transfer sites. PLoS One 8:e67465

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