Modifications are found in all tRNA species from all organisms. A typical yeast tRNA has two to three modifications in and around the anticodon loop, where decoding takes place, and another ten modifications in the main body of the tRNA, remote from the anticodon loop. These modifications have crucial roles, many of which have emerged in the last few years, but many of which are still poorly understood. Work in this lab focuses on two major aspects of the biology of tRNA and its modifications in the yeast Saccharomyces cerevisiae. One project addresses the biology and mechanisms of a rapid tRNA decay quality control pathway that acts widely on specific mature tRNAs that lack one or more of several different modifications, or that have mutations that perturb structure in the acceptor and/or T-stem. A second project addresses specific modifications in the anticodon loop that have important functions in translation, with a major effort to understand the biology and specificity of Trm7, which is required for 2'-O-methylation of residues C32 and N34 to make Cm32 and Nm34 in tRNAPhe, tRNATrp and tRNALeu(UAA). S. cerevisiae trm7-? mutants have a severe growth defect, which this lab recently showed is due to lack of sufficient functional tRNAPhe. Furthermore, this lab also provided evidence for a complex circuitry of anticodon loop modification of tRNAPhe, in which Trm7 interacts with YMR259c (now named Trm732) for formation of Cm32, and with Rtt10 (now named Trm734) for formation of Gm34, which in turn drives modification of 1-methylguanosine to yW (wyebutosine). This lab has speculated that the Trm7:Trm732 and Trm7:Trm734 complexes and the unusual circuitry of anticodon loop modification of tRNAPhe are widely conserved and important in eukaryotes. In support of this speculation, the putative human TRM7 homolog FTSJ1 is strongly associated with non-syndromic X-linked mental retardation, and the corresponding TRM7 homolog from Schizosaccharomyces pombe is reported to be essential. Furthermore, almost all characterized eukaryotic tRNAPhe species have Cm32 and Gm34, orthologs of Trm7, Trm732, and Trm734 are found in almost all eukaryotes, and the human Trm7 and Trm732 orthologs function in S. cerevisiae. Based on these observations, the main goal of this proposal is to determine if the complex circuitry for anticodon loop modification of tRNAPhe is conserved and important in eukaryotes. This goal will be addressed by (A.) Defining requirements in S. cerevisiae for interaction and activity of Trm732 and Trm734 with Trm7;and (B.) Determining if the complex circuitry and importance of Trm7 modification of the anticodon loop is conserved in other eukaryotes. By examining the parameters that define the Trm7 interactions in S. cerevisiae, and by defining the Trm7 complexes and their target tRNAs in S. pombe, Drosophila, and humans, we will acquire sophisticated understanding of this critically important set of tRNAPhe modifications in the anticodon loop.

Public Health Relevance

Synthesis of all proteins in cells requires proper decoding of messenger RNA by transfer RNA (tRNA) during translation by the ribosome, which requires modifications in the anticodon loop of the tRNAs. A number of human health conditions and diseases are associated with defects in tRNA modifications, including those addressed in this project, which is directed toward study of two modifications in the anticodon loop that result in poor growth if not present in the model eukaryotic organism Saccharomyces cerevisiae, and are associated with mental retardation if not present in humans. The methyltransferase responsible for these modifications forms two different complexes in S. cerevisiae, one for each of the modifications, and these modifications are in turn required for a third modification in the anticodon loop of the tRNA by another protein;the object of this proposal is to determine if this circuitry of complexes and modifications is conserved in several other eukaryotes, including two other well studied model organisms and humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM052347-18S1
Application #
8573155
Study Section
Special Emphasis Panel (ZGM1-CBB-0 (MI))
Program Officer
Bender, Michael T
Project Start
1995-05-01
Project End
2015-12-31
Budget Start
2013-01-01
Budget End
2013-12-31
Support Year
18
Fiscal Year
2013
Total Cost
$47,969
Indirect Cost
$16,719
Name
University of Rochester
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
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Preston, Melanie A; D'Silva, Sonia; Kon, Yoshiko et al. (2013) tRNAHis 5-methylcytidine levels increase in response to several growth arrest conditions in Saccharomyces cerevisiae. RNA 19:243-56
Whipple, Joseph M; Lane, Elizabeth A; Chernyakov, Irina et al. (2011) The yeast rapid tRNA decay pathway primarily monitors the structural integrity of the acceptor and T-stems of mature tRNA. Genes Dev 25:1173-84
Wilusz, Jeremy E; Whipple, Joseph M; Phizicky, Eric M et al. (2011) tRNAs marked with CCACCA are targeted for degradation. Science 334:817-21
D'Silva, Sonia; Haider, Steffen J; Phizicky, Eric M (2011) A domain of the actin binding protein Abp140 is the yeast methyltransferase responsible for 3-methylcytidine modification in the tRNA anti-codon loop. RNA 17:1100-10
Murthi, Athulaprabha; Shaheen, Hussam H; Huang, Hsiao-Yun et al. (2010) Regulation of tRNA bidirectional nuclear-cytoplasmic trafficking in Saccharomyces cerevisiae. Mol Biol Cell 21:639-49
Phizicky, Eric M; Hopper, Anita K (2010) tRNA biology charges to the front. Genes Dev 24:1832-60
Preston, Melanie A; Phizicky, Eric M (2010) The requirement for the highly conserved G-1 residue of Saccharomyces cerevisiae tRNAHis can be circumvented by overexpression of tRNAHis and its synthetase. RNA 16:1068-77
Kotelawala, Lakmal; Grayhack, Elizabeth J; Phizicky, Eric M (2008) Identification of yeast tRNA Um(44) 2'-O-methyltransferase (Trm44) and demonstration of a Trm44 role in sustaining levels of specific tRNA(Ser) species. RNA 14:158-69

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