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.
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.
|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 Î¨38 and Î¨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|
|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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