tRNAs are highly evolved in all organisms for specific recognition by cognate tRNA synthetases, high fidelity decoding, efficient use in translation, and high stability. The ubiquitous tRNA modifications are highly conserved in eukaryotes, and many have crucial roles in the yeast Saccharomyces cerevisiae and in human health. Modifications in the tRNA body (outside the anticodon loop) are crucial for tRNA stability in yeast, and associated with several neurological disorders in humans. We study the rapid tRNA decay (RTD) pathway in S. cerevisiae, which targets a subset of mature tRNAs lacking any of several body modifications, due to exposure of the 5' end to the 5'-3' exonucleases Rat1 and Xrn1. RTD also frequently occurs in tRNA variants with destabilizing mutations exposing the 5' end, and is inhibited in met22? mutants due to increased levels of adenosine 3',5' bis-phosphate (pAp) and its inhibition of Rat1 and Xrn1. Little is known about RTD or the biology of body modifications in any other eukaryote. To address this, we are studying these processes in the fission yeast Schizosaccharomyces pombe because of its ~600 million years evolutionary distance from S. cerevisiae, and because of its facile genetics and molecular biology. We have recently uncovered an unusual decay pathway in S. cerevisiae in which pre-tRNAs are degraded in the cytoplasm by a pathway regulated by Met22. This Met22-regulated pre-tRNA decay (MPD) pathway is independent of RTD, because unlike classical RTD, it does not require the Rat1 or Xrn1 exonucleases and does not act on mature tRNA, and it is novel because it is also independent of the nuclear surveillance tRNA decay pathway, which acts in the nucleus on pre-tRNAs through Trf4, RRP6 and the nuclear exosome. Rather, MPD occurs on unspliced pre-tRNA that accumulates in the cytoplasm due to impaired intron-exon structure. We also study modifications in the anticodon loop, due to their importance in translation, with a focus on Trm7, which 2?-O-methylates N32 and N34 in the anticodon loop of certain tRNAs. S. cerevisiae and S. pombe trm7 mutants have severe growth defects, while humans with mutations have intellectual disability. Our prior results showed that the growth defect of S. cerevisiae and S. pombe trm7 mutants was due to reduced function, but not reduced amounts, of tRNAPhe. We recently discovered an unusual property of S. cerevisiae and S. pombe trm7? mutants: each mutant robustly activates the general amino acid control (GAAC) response, which massively reprograms gene expression in all eukaryotes due to uncharged tRNA sensed by Gcn2 kinase, but trm7? mutants do not exhibit a detectable tRNA charging defect. To follow up, we will: 1) Examine similarities and differences in the RTD pathway and body modification biology in S. pombe 2) Define how Met22-regulated pre-tRNA decay of anticodon stem variants occurs in S. cerevisiae 3) Define how trm7? mutants activate the GAAC pathway and how Trm7 recognizes tRNAs.

Public Health Relevance

Transfer RNAs (tRNAs) are critical for decoding messenger RNA during translation by the ribosome, where they insert their charged amino acid into the end of the growing peptide chain. The numerous tRNA modifications found in all organisms are critical for function and stability, are highly conserved in the yeast Saccharomyces cerevisiae and in humans, and are required to prevent poor growth in yeast and human neurological disorders and mitochondrial diseases. This project is directed toward study of a critical tRNA decay pathway in S. cerevisiae and the evolutionarily distant yeast Schizosaccharomyces pombe, the analysis of a novel tRNA decay pathway in S. cerevisiae that monitors the quality of tRNA near the tRNA decoding region, and in understanding how lack of a modification near the tRNA decoding region can activate a major control pathway in both S. cerevisiae and S. pombe in the absence of the uncharged tRNA (lacking an amino acid) that is normally required to activate this pathway.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics A Study Section (MGA)
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Bender, Michael T
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University of Rochester
School of Medicine & Dentistry
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Zimmerman, Stephanie M; Kon, Yoshiko; Hauke, Alayna C et al. (2018) Conditional accumulation of toxic tRNAs to cause amino acid misincorporation. Nucleic Acids Res 46:7831-7843
Han, Lu; Guy, Michael P; Kon, Yoshiko et al. (2018) Lack of 2'-O-methylation in the tRNA anticodon loop of two phylogenetically distant yeast species activates the general amino acid control pathway. PLoS Genet 14:e1007288
Payea, Matthew J; Sloma, Michael F; Kon, Yoshiko et al. (2018) Widespread temperature sensitivity and tRNA decay due to mutations in a yeast tRNA. RNA 24:410-422
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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
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
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
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

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