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      N 7-Methylguanine at position 46 (m 7G46) in tRNA from Thermus thermophilus is required for cell viability at high temperatures through a tRNA modification network

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          Abstract

          N 7-methylguanine at position 46 (m 7G46) in tRNA is produced by tRNA (m 7G46) methyltransferase (TrmB). To clarify the role of this modification, we made a trmB gene disruptant ( ΔtrmB) of Thermus thermophilus, an extreme thermophilic eubacterium. The absence of TrmB activity in cell extract from the ΔtrmB strain and the lack of the m 7G46 modification in tRNA Phe were confirmed by enzyme assay, nucleoside analysis and RNA sequencing. When the ΔtrmB strain was cultured at high temperatures, several modified nucleotides in tRNA were hypo-modified in addition to the lack of the m 7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m 1G37, suggesting that the m 7G46 positively affects their formations. Although the lack of the m 7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNA Phe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNA Phe and tRNA Ile. 35S-Met incorporation into proteins revealed that protein synthesis in ΔtrmB cells is depressed above 70°C. At 80°C, the ΔtrmB strain exhibits a severe growth defect. Thus, the m 7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m 7G46 modification supports introduction of other modifications.

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          Most cited references55

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          Compilation of tRNA sequences and sequences of tRNA genes.

          Sequences of 3279 sequences of tRNA genes and tRNAs published up to December 1996 are included in the compilation. Alignment of the sequences, which is most compatible with the tRNA phylogeny and known three-dimensional structures of tRNA, is used. Sequences and references are available under http://www.uni-bayreuth. de/departments/biochemie/trna/
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            Rapid tRNA decay can result from lack of nonessential modifications.

            The biological role of many nonessential tRNA modifications outside of the anticodon remains elusive despite their evolutionary conservation. We show here that m7G46 methyltransferase Trm8p/Trm82p acts as a hub of synthetic interactions with several tRNA modification enzymes, resulting in temperature-sensitive growth. Analysis of three double mutants indicates reduced levels of tRNA(Val(AAC)), consistent with a role of the corresponding modifications in maintenance of tRNA levels. Detailed examination of a trm8-delta trm4-delta double mutant demonstrates rapid degradation of preexisting tRNA(Val(AAC)) accompanied by its de-aminoacylation. Multiple copies of tRNA(Val(AAC)) suppress the trm8-delta trm4-delta growth defect, directly implicating this tRNA in the phenotype. These results define a rapid tRNA degradation (RTD) pathway that is independent of the TRF4/RRP6-dependent nuclear surveillance pathway. The degradation of an endogenous tRNA species at a rate typical of mRNA decay demonstrates a critical role of nonessential modifications for tRNA stability and cell survival.
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              Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA.

              7-methylguanosine (m7G) modification of tRNA occurs widely in eukaryotes and bacteria, is nearly always found at position 46, and is one of the few modifications that confers a positive charge to the base. Screening of a Saccharomyces cerevisiae genomic library of purified GST-ORF fusion proteins reveals two previously uncharacterized proteins that copurify with m7G methyltransferase activity on pre-tRNA(Phe). ORF YDL201w encodes Trm8, a protein that is highly conserved in prokaryotes and eukaryotes and that contains an S-adenosylmethionine binding domain. ORF YDR165w encodes Trm82, a less highly conserved protein containing putative WD40 repeats, which are often implicated in macromolecular interactions. Neither protein has significant sequence similarity to yeast Abd1, which catalyzes m7G modification of the 5' cap of mRNA, other than the methyltransferase motif shared by Trm8 and Abd1. Several lines of evidence indicate that both Trm8 and Trm82 proteins are required for tRNA m7G-methyltransferase activity: Extracts derived from strains lacking either gene have undetectable m7G methyltransferase activity, RNA from strains lacking either gene have much reduced m7G, and coexpression of both proteins is required to overproduce activity. Aniline cleavage mapping shows that Trm8/Trm82 proteins modify pre-tRNAPhe at G46, the site that is modified in vivo. Trm8 and Trm82 proteins form a complex, as affinity purification of Trm8 protein causes copurification of Trm82 protein in approximate equimolar yield. This functional two-protein family appears to be retained in eukaryotes, as expression of both corresponding human proteins, METTL1 and WDR4, is required for m7G-methyltransferase activity.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                January 2010
                24 November 2009
                24 November 2009
                : 38
                : 3
                : 942-957
                Affiliations
                1Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, 2Department of Biomolecular Science, Faculty of Engineering, Gifu University, Yanagido 1-1, Gifu, Gifu 501-1193, 3Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Katsura, Nishikyo-ku, Kyoto, Kyoto 615-8510, 4Venture Business Laboratory, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577 and 5RIKEN SPring-8 Center, Kouto 1-1-1, Sayo-cho, Sayo-gun, Hyougo 679-5148, Japan
                Author notes
                *To whom correspondence should be addressed. Tel: +81 89 927 8548; Fax: +81 89 927 9941; Email: hori@ 123456eng.ehime-u.ac.jp
                Article
                gkp1059
                10.1093/nar/gkp1059
                2817472
                19934251
                ab7023cc-1797-4616-8deb-4af0a2227f13
                © The Author(s) 2009. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 9 September 2009
                : 24 October 2009
                : 26 October 2009
                Categories
                Nucleic Acid Enzymes

                Genetics
                Genetics

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