The sequence, structure, and modifications of tRNAs are exquisitely tuned for high fidelity charging and decoding, for efficient use in translation, and for high stability. One major goal of this project is to define the scope and specificity of a major quality control pathway in the yeast Saccharomyces cerevisiae that targets mature tRNAs lacking certain modifications for rapid tRNA decay (RTD), resulting in growth defects. We previously found that RTD acts on tRNAs lacking one or more of several modifications, is mediated by the 5'-3' exonucleases Rat1 and Xrn1, is inhibited by a met22? mutation, and acts on only a subset of tRNA species lacking the particular modifications. Understanding the specificity of this pathway is important because recent results in the field show that RTD is conserved in HeLa cells and that reduced tRNA levels occur in certain mouse modification mutants and in a number of mitochondrial diseases associated with tRNA mutations. Prior investigation of the tRNASer family showed that RTD occurs on fully modified and hypomodified tRNAs with destabilized acceptor and T-stems, resulting in increased exposure of the 5' ends, consistent with degradation by 5'-3' exonucleases. However, our recent high throughput analysis of tRNATyr function (using the SUP4oc nonsense suppressor) provides evidence that RTD is also triggered by a destabilized anticodon stem-loop (ASL), suggesting a new mechanism to engage RTD. In addition, we find evidence for an unexpected tRNA decay pathway that occurs on mature tRNAs in the absence of RTD. A second major project focuses on the biology of selected modifications associated with growth defects in yeast and with human disease. Defects in any of several different human modification genes are associated with intellectual disability, microcephaly, or familial dysautonomia. A major current goal is to understand the severe growth defect of cells lacking Trm7, which is required for 2'-O-methyation of C32 and N34 of 3 tRNA species. The human TRM7 homolog FTSJ1 is linked to non-syndromic X-linked intellectual disability (NSXLID). We found that the S. cerevisiae trm7? growth defect is due to lack of functional tRNAPhe and that tRNAPhe undergoes an intricate set of modifications in which Trm7 works with Trm732 and with Trm734 to catalyze Cm32 and Gm34 modifications, which then promote wybutosine (yW) formation at m1G37. We also found that this circuitry is retained and important in the distantly related yeast Schizosaccharomyces pombe. To follow up, we propose: (1) To define the mechanisms by which tRNA is recognized and degraded in yeast. (2) To define the mechanisms by which the RTD pathway is regulated, and (3) To determine the roles of selected modification genes implicated in human disease, with a focus on analysis of cell lines from NSXLID patients with FTSJ1 lesions, and on the roles and specificity of yeast Trm7 and its partner proteins.

Public Health Relevance

Synthesis of all proteins requires decoding of messenger RNA by transfer RNA (tRNA) during translation by the ribosome. All tRNAs are highly stable and highly modified, and the modifications found in eukaryotes are almost all conserved in the model eukaryotic organism Saccharomyces cerevisiae and in humans. This project is directed toward study of the role of tRNA modifications and of a prominent tRNA decay pathway in yeast, in which it is possible to study biological function in great detail, and in the roles of two human modification genes linked to intellectual disability or microcephaly.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM052347-23
Application #
9405012
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1995-05-01
Project End
2019-12-31
Budget Start
2018-01-01
Budget End
2018-12-31
Support Year
23
Fiscal Year
2018
Total Cost
Indirect Cost
Name
University of Rochester
Department
Biochemistry
Type
School of Medicine & Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Han, Lu; Marcus, Erin; D'Silva, Sonia et al. (2017) S. cerevisiae Trm140 has two recognition modes for 3-methylcytidine modification of the anticodon loop of tRNA substrates. RNA 23:406-419
Hrabeta-Robinson, Eva; Marcus, Erin; Cozen, Aaron E et al. (2017) High-Throughput Small RNA Sequencing Enhanced by AlkB-Facilitated RNA de-Methylation (ARM-Seq). Methods Mol Biol 1562:231-243
Shaheen, Ranad; Han, Lu; Faqeih, Eissa et al. (2016) A homozygous truncating mutation in PUS3 expands the role of tRNA modification in normal cognition. Hum Genet 135:707-13
Guy, Michael P; Shaw, Marie; Weiner, Catherine L et al. (2015) Defects in tRNA Anticodon Loop 2'-O-Methylation Are Implicated in Nonsyndromic X-Linked Intellectual Disability due to Mutations in FTSJ1. Hum Mutat 36:1176-87
Payea, Matthew J; Guy, Michael P; Phizicky, Eric M (2015) Methodology for the High-Throughput Identification and Characterization of tRNA Variants That Are Substrates for a tRNA Decay Pathway. Methods Enzymol 560:1-17
Cozen, Aaron E; Quartley, Erin; Holmes, Andrew D et al. (2015) ARM-seq: AlkB-facilitated RNA methylation sequencing reveals a complex landscape of modified tRNA fragments. Nat Methods 12:879-84
Han, Lu; Kon, Yoshiko; Phizicky, Eric M (2015) Functional importance of ?38 and ?39 in distinct tRNAs, amplified for tRNAGln(UUG) by unexpected temperature sensitivity of the s2U modification in yeast. RNA 21:188-201
Guy, Michael P; Phizicky, Eric M (2015) Conservation of an intricate circuit for crucial modifications of the tRNAPhe anticodon loop in eukaryotes. RNA 21:61-74
Shaheen, Ranad; Abdel-Salam, Ghada M H; Guy, Michael P et al. (2015) Mutation in WDR4 impairs tRNA m(7)G46 methylation and causes a distinct form of microcephalic primordial dwarfism. Genome Biol 16:210
Phizicky, Eric M; Hopper, Anita K (2015) tRNA processing, modification, and subcellular dynamics: past, present, and future. RNA 21:483-5

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