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

PROJECT NARRATIVE 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM052347-17
Application #
8250542
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Bender, Michael T
Project Start
1995-05-01
Project End
2015-12-31
Budget Start
2012-04-01
Budget End
2012-12-31
Support Year
17
Fiscal Year
2012
Total Cost
$316,725
Indirect Cost
$111,725
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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
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
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
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
Han, Lu; Kon, Yoshiko; Phizicky, Eric M (2015) Functional importance of Ψ38 and Ψ39 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
Pop, Cristina; Rouskin, Silvi; Ingolia, Nicholas T et al. (2014) Causal signals between codon bias, mRNA structure, and the efficiency of translation and elongation. Mol Syst Biol 10:770
Zhang, Xiaoju; Walker, Ross C; Phizicky, Eric M et al. (2014) Influence of Sequence and Covalent Modifications on Yeast tRNA Dynamics. J Chem Theory Comput 10:3473-3483

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