The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA.

The methylation of pseudouridine (Ψ) at position 54 of tRNA, producing m(1)Ψ, is a hallmark of many archaeal species, but the specific methylase involved in the formation of this modification had yet to be characterized. A comparative genomics analysis had previously identified COG1901 (DUF358), part of the SPOUT superfamily, as a candidate for this missing methylase family. To test this prediction, the COG1901 encoding gene, HVO_1989, was deleted from the Haloferax volcanii genome. Analyses of modified base contents indicated that while m(1)Ψ was present in tRNA extracted from the wild-type strain, it was absent from tRNA extracted from the mutant strain. Expression of the gene encoding COG1901 from Halobacterium sp. NRC-1, VNG1980C, complemented the m(1)Ψ minus phenotype of the ΔHVO_1989 strain. This in vivo validation was extended with in vitro tests. Using the COG1901 recombinant enzyme from Methanocaldococcus jannaschii (Mj1640), purified enzyme Pus10 from M. jannaschii and full-size tRNA transcripts or TΨ-arm (17-mer) fragments as substrates, the sequential pathway of m(1)Ψ54 formation in Archaea was reconstituted. The methylation reaction is AdoMet dependent. The efficiency of the methylase reaction depended on the identity of the residue at position 55 of the TΨ-loop. The presence of Ψ55 allowed the efficient conversion of Ψ54 to m(1)Ψ54, whereas in the presence of C55, the reaction was rather inefficient and no methylation reaction occurred if a purine was present at this position. These results led to renaming the Archaeal COG1901 members as TrmY proteins.

[1]  H. Grosjean.  Nucleic Acids Are Not Boring Long Polymers of Only Four Types of Nucleotides: A Guided Tour , 2013 .

[2]  Yuri Motorin,et al.  RNA nucleotide methylation , 2011, Wiley interdisciplinary reviews. RNA.

[3]  Joe R. Cannon,et al.  Structural insight into the functional mechanism of Nep1/Emg1 N1-specific pseudouridine methyltransferase in ribosome biogenesis , 2010, Nucleic acids research.

[4]  Jef Rozenski,et al.  The RNA modification database, RNAMDB: 2011 update , 2010, Nucleic Acids Res..

[5]  K. Entian,et al.  The Bowen–Conradi syndrome protein Nep1 (Emg1) has a dual role in eukaryotic ribosome biogenesis, as an essential assembly factor and in the methylation of Ψ1191 in yeast 18S rRNA , 2010, Nucleic acids research.

[6]  S. Douthwaite,et al.  Specificity shifts in the rRNA and tRNA nucleotide targets of archaeal and bacterial m5U methyltransferases. , 2011, RNA.

[7]  Ian K. Blaby,et al.  Towards a Systems Approach in the Genetic Analysis of Archaea: Accelerating Mutant Construction and Phenotypic Analysis in Haloferax volcanii , 2010, Archaea.

[8]  Y. Yuan,et al.  Crystal structure of Mj1640/DUF358 protein reveals a putative SPOUT-class RNA methyltransferase. , 2010, Journal of molecular cell biology.

[9]  V. de Crécy-Lagard,et al.  Biosynthesis of wyosine derivatives in tRNA: an ancient and highly diverse pathway in Archaea. , 2010, Molecular biology and evolution.

[10]  K. Entian,et al.  The ribosome assembly factor Nep1 responsible for Bowen–Conradi syndrome is a pseudouridine-N1-specific methyltransferase , 2010, Nucleic acids research.

[11]  Joanna M. Kasprzak,et al.  MODOMICS: a database of RNA modification pathways. 2008 update , 2008, Nucleic Acids Research.

[12]  Priyatansh Gurha,et al.  Archaeal Pus10 proteins can produce both pseudouridine 54 and 55 in tRNA. , 2008, RNA.

[13]  Henri Grosjean,et al.  RNomics and Modomics in the halophilic archaea Haloferax volcanii: identification of RNA modification genes , 2008, BMC Genomics.

[14]  Priyatansh Gurha,et al.  Box C/D RNA-Guided 2′-O Methylations and the Intron of tRNATrp Are Not Essential for the Viability of Haloferax volcanii , 2008, Journal of bacteriology.

[15]  B. Golinelli‐Pimpaneau,et al.  The crystal structure of Pyrococcus abyssi tRNA (uracil-54, C5)-methyltransferase provides insights into its tRNA specificity , 2008, Nucleic acids research.

[16]  R. Stroud,et al.  Structure of a TrmA–RNA complex: A consensus RNA fold contributes to substrate selectivity and catalysis in m5U methyltransferases , 2008, Proceedings of the National Academy of Sciences.

[17]  K. Entian,et al.  The crystal structure of Nep1 reveals an extended SPOUT-class methyltransferase fold and a pre-organized SAM-binding site , 2008, Nucleic acids research.

[18]  B. Golinelli‐Pimpaneau,et al.  Acquisition of a bacterial RumA‐type tRNA(uracil‐54, C5)‐methyltransferase by Archaea through an ancient horizontal gene transfer , 2007, Molecular microbiology.

[19]  D. Tollervey,et al.  The yeast ribosome synthesis factor Emg1 is a novel member of the superfamily of alpha/beta knot fold methyltransferases , 2007, Nucleic acids research.

[20]  Active suppressor tRNAs with a double helix between the D- and T-loops. , 2007, Journal of molecular biology.

[21]  Priyatansh Gurha,et al.  Differential Roles of Archaeal Box H/ACA Proteins in Guide RNA-Dependent and Independent Pseudouridine Formation , 2007, RNA biology.

[22]  C. Woese,et al.  Unusual modification patterns in the transfer ribonucleic acids of archaebacteria , 1980, Current Microbiology.

[23]  L. Droogmans,et al.  Detection of enzymatic activity of transfer RNA modification enzymes using radiolabeled tRNA substrates. , 2007, Methods in enzymology.

[24]  Christiane Branlant,et al.  Identification of modified residues in RNAs by reverse transcription-based methods. , 2007, Methods in enzymology.

[25]  Janusz M. Bujnicki,et al.  Structural and evolutionary bioinformatics of the SPOUT superfamily of methyltransferases , 2007, BMC Bioinformatics.

[26]  K. Entian,et al.  Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p , 2006, Molecular Genetics and Genomics.

[27]  Marcin Feder,et al.  MODOMICS: a database of RNA modification pathways , 2005, Nucleic Acids Res..

[28]  Naryttza N. Diaz,et al.  The Subsystems Approach to Genome Annotation and its Use in the Project to Annotate 1000 Genomes , 2005, Nucleic acids research.

[29]  Robert M. Stroud,et al.  A Unique RNA Fold in the RumA-RNA-Cofactor Ternary Complex Contributes to Substrate Selectivity and Enzymatic Function , 2005, Cell.

[30]  Robert C. Edgar,et al.  MUSCLE: multiple sequence alignment with high accuracy and high throughput. , 2004, Nucleic acids research.

[31]  R. G. Lloyd,et al.  Development of Additional Selectable Markers for the Halophilic Archaeon Haloferax volcanii Based on the leuB and trpA Genes , 2004, Applied and Environmental Microbiology.

[32]  R. Cavicchioli,et al.  Influence of Temperature on tRNA Modification in Archaea: Methanococcoides burtonii (Optimum Growth Temperature [Topt], 23°C) and Stetteria hydrogenophila (Topt, 95°C) , 2003 .

[33]  Darren A. Natale,et al.  The COG database: an updated version includes eukaryotes , 2003, BMC Bioinformatics.

[34]  John P. Huelsenbeck,et al.  MrBayes 3: Bayesian phylogenetic inference under mixed models , 2003, Bioinform..

[35]  Rodrigo Lopez,et al.  Multiple sequence alignment with the Clustal series of programs , 2003, Nucleic Acids Res..

[36]  I. Stansfield,et al.  Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance. , 2002, Microbiology.

[37]  T. Henkin.  Faculty Opinions recommendation of tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features. , 2002 .

[38]  Henri Grosjean,et al.  tRNomics: analysis of tRNA genes from 50 genomes of Eukarya, Archaea, and Bacteria reveals anticodon-sparing strategies and domain-specific features. , 2002, RNA.

[39]  J. Ofengand,et al.  Mapping pseudouridines in RNA molecules. , 2001, Methods.

[40]  J. Ofengand,et al.  Deletion of the Escherichia coli pseudouridine synthase gene truB blocks formation of pseudouridine 55 in tRNA in vivo, does not affect exponential growth, but confers a strong selective disadvantage in competition with wild-type cells. , 2000, RNA.

[41]  J. Kowalak,et al.  Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii * , 2000, The Journal of Biological Chemistry.

[42]  Y. Motorin,et al.  Transfer RNA modification enzymes from Pyrococcus furiosus: detection of the enzymatic activities in vitro. , 1999, Nucleic acids research.

[43]  Andrei V Bakin,et al.  Mapping of pseudouridine residues in RNA to nucleotide resolution. , 1998, Methods in molecular biology.

[44]  C. Florentz,et al.  Major identity determinants for enzymatic formation of ribothymidine and pseudouridine in the T psi-loop of yeast tRNAs. , 1997, Journal of molecular biology.

[45]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[46]  Gapped BLAST and PSI-BLAST: A new , 1997 .

[47]  D. Santi,et al.  Recognition of the T-arm of tRNA by tRNA (m5U54)-methyltransferase is not sequence specific. , 1996, Biochemistry.

[48]  R. Giegé,et al.  Enzymatic formation of modified nucleosides in tRNA: dependence on tRNA architecture. , 1996, Journal of molecular biology.

[49]  H. Grosjean,et al.  A novel enzymatic pathway leading to 1-methylinosine modification in Haloferax volcanii tRNA. , 1995, Nucleic acids research.

[50]  J. Kowalak,et al.  The role of posttranscriptional modification in stabilization of transfer RNA from hyperthermophiles. , 1994, Biochemistry.

[51]  C. G. Edmonds,et al.  Posttranscriptional modification of tRNA in thermophilic archaea (Archaebacteria) , 1991, Journal of bacteriology.

[52]  P. Crain,et al.  Preparation and enzymatic hydrolysis of DNA and RNA for mass spectrometry. , 1990, Methods in enzymology.

[53]  E. Westhof,et al.  Importance of conserved residues for the conformation of the T-loop in tRNAs. , 1987, Journal of biomolecular structure & dynamics.

[54]  Ramesh C. Gupta.  Transfer RNAs of Halobacterium volcanii: Sequences of five leucine and three serine tRNAs* , 1986 .

[55]  R. Cedergren,et al.  Halobacterium cutirubrum tRNA sequences , 1985 .

[56]  C. G. Edmonds,et al.  Thermospray liquid chromatography-mass spectrometry of nucleosides and of enzymatic hydrolysates of nucleic acids. , 1985, Nucleic acids research.

[57]  R. Cedergren,et al.  Glycine and asparagme tRNA sequences from the archaebacteriuin, Methanobacterium thermoautotrophicum , 1984 .

[58]  R. Gupta.  Halobacterium volcanii tRNAs. Identification of 41 tRNAs covering all amino acids, and the sequences of 33 class I tRNAs. , 1984, The Journal of biological chemistry.

[59]  R. Cedergren,et al.  Sequences of halobacterial tRNAs and the paucity of U in the first position of their anticodons. , 1983, Nucleic acids research.

[60]  M. Ihara,et al.  Initiator tRNAs from archaebacteria show common unique sequence characteristics , 1982, Nature.

[61]  C. Woese,et al.  Structure of a modified nucleoside in archaebacterial tRNA which replaces ribosylthymine. 1-Methylpseudouridine. , 1982, The Journal of biological chemistry.

[62]  R. T. Walker,et al.  The nucleotide sequence of the tRNAMMet from the archaebacterium Thermoplasma acidophilum. , 1981, Nucleic acids research.

[63]  J. Hearst,et al.  A sequence from Drosophila melanogaster 18S rRNA bearing the conserved hypermodified nucleoside am psi: analysis by reverse transcription and high-performance liquid chromatography. , 1981, Nucleic acids research.

[64]  E. D. De Robertis,et al.  RNA processing in microinjected Xenopus oocytes. Sequential addition of base modifications in the spliced transfer RNA. , 1981, Journal of molecular biology.

[65]  G. Björk,et al.  Cloning and restriction mapping of the trmA gene coding for transfer ribonucleic acid (5-methyluridine)-methyltransferase in Escherichia coli K-12 , 1980, Journal of bacteriology.

[66]  J. Goddard.  Transfer RNA , 1980, Nature.

[67]  Portland Press Ltd.  Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid , 1978 .

[68]  A. Best.  Composition and Characterization of tRNA from Methanococcus vannielii , 1978, Journal of bacteriology.

[69]  R. Planta,et al.  Biosynthesis of a hypermodified nucleotide in Saccharomyces carlsbergensis 17S and HeLa-cell 18S ribosomal ribonucleic acid. , 1978, The Biochemical journal.