The structure, sequence, and extensive modifications of tRNAs make them remarkably well designed for specific recognition by synthetases, and uniform use during translation. tRNA modifications are highly conserved among species, and have a number of roles in cell function and human health. However, our understanding of the precise roles of modifications has been elusive in yeast, as studied here, and in humans. One long term goal of this project is to define a tRNA quality control pathway that monitors the integrity of mature tRNA. The lab previously showed that yeast trm8- trm4- mutants (which lack 7-methylguanosine and 5-methylcytidine) are temperature sensitive due to rapid tRNA decay (RTD) of mature tRNAVal(AAC) by an unknown pathway. Recent work shows that RTD is mediated by the 5'-3'exonucleases Rat1 and Xrn1, and by Met22, and degrades different specific mature tRNA species in strains lacking different modifications, but not other tRNA species lacking the same modifications. Subsequent analysis indicates that specific tRNAs are targeted for RTD because their acceptor and T-stems are less stable and cause increased exposure of their 5'end, that modifications impact RTD indirectly through their effect on tertiary structure, and that Xrn1 selectively degrades RTD substrate tRNAs in vitro. Moreover, preliminary results implicate components of the translation machinery in RTD, since RTD is affected by the elongation factor EF-1A, which normally binds charged tRNA for delivery to the ribosome, and by Bud27, which affects translation initiation, and is reported to bind EF-1A . A second long term goal is to understand the biology of modifications around the anticodon loop to clarify their effects on translation. Current work is focusing on 2'-O-methylation of C32 (Cm32) and N34, which occurs on three tRNAs and requires Trm7, and on 3-methylcytidine modification of C32 (m3C32), which occurs on six other tRNA species, although it is unclear how these tRNAs are distinguished for each modification. Recent results showed that a domain of the actin binding protein Abp140 (Trm140) is required for formation of m3C32 for each of the six tRNA species, and that trm140- mutants are modestly translation defective. Preliminary work also suggests that the severe trm7- growth defect is caused by failure to modify one particular tRNA at C32, and implicates a previously unrecognized second subunit in Trm7 activity. This may have implications in human health since lack of Trm7 is associated with mental retardation. To follow up on these results, four aims are proposed: (1) To determine the mechanism by which the RTD pathway recognizes and degrades tRNA substrates (2) To determine the roles of anticodon loop modifications catalyzed by Trm7 and Trm140 (3) To define substrate specificity for m3C32, Cm32, and Nm34 modifications by Trm7 and Trm140 and (4) To use genomic methods to probe limits of tRNA function, RTD substrates and conditional mutants.

Public Health Relevance

Synthesis of all proteins in cells requires decoding of messenger RNA by transfer RNA (tRNA) during translation by the ribosome. tRNAs are highly modified, and these modifications are highly conserved in all organisms, including humans;moreover, a number of human health conditions and diseases are associated with defects in tRNA modifications. This project is directed toward study of the role of tRNA modifications in the model eukaryotic organism Saccharomyces cerevisiae, in which it is possible to study biological function in great detail, for possible subsequent application to human conditions.

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
Schools of 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
Han, Lu; Phizicky, Eric M (2018) A rationale for tRNA modification circuits in the anticodon loop. RNA 24:1277-1284
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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