Publications

• Biosynthesis of wyosine derivatives in tRNA: an ancient and highly diverse pathway in Archaea.

  de Crecy-Lagard V., Brochier-Armanet C., Urbonavicius J., Fernandez B., Phillips G., Lyons B., Noma A., Alvarez S., Droogmans L., Armengaud J., Grosjean H.

  Wyosine (imG) and its derivatives such as wybutosine (yW) are found at position 37 of phenylalanine-specific transfer RNA (tRNA(Phe)), 3' adjacent to the anticodon in Eucarya and Archaea. In Saccharomyces cerevisiae, formation of yW requires five enzymes acting in a strictly sequential order: Trm5 ... >> More

  Wyosine (imG) and its derivatives such as wybutosine (yW) are found at position 37 of phenylalanine-specific transfer RNA (tRNA(Phe)), 3' adjacent to the anticodon in Eucarya and Archaea. In Saccharomyces cerevisiae, formation of yW requires five enzymes acting in a strictly sequential order: Trm5, Tyw1, Tyw2, Tyw3, and Tyw4. Archaea contain wyosine derivatives, but their diversity is greater than in eukaryotes and the corresponding biosynthesis pathways still unknown. To identify these pathways, we analyzed the phylogenetic distribution of homologues of the yeast wybutosine biosynthesis proteins in 62 archaeal genomes and proposed a scenario for the origin and evolution of wyosine derivatives biosynthesis in Archaea that was partly experimentally validated. The key observations were 1) that four of the five wybutosine biosynthetic enzymes are ancient and may have been present in the last common ancestor of Archaea and Eucarya, 2) that the variations in the distribution pattern of biosynthesis enzymes reflect the diversity of the wyosine derivatives found in different Archaea. We also identified 7-aminocarboxypropyl-demethylwyosine (yW-86) and its N4-methyl derivative (yW-72) as final products in tRNAs of several Archaea when these were previously thought to be only intermediates of the eukaryotic pathway. We confirmed that isowyosine (imG2) and 7-methylwyosine (mimG) are two archaeal-specific guanosine-37 derivatives found in tRNA of both Euryarchaeota and Crenarchaeota. Finally, we proposed that the duplication of the trm5 gene in some Archaea led to a change in function from N1 methylation of guanosine to C7 methylation of 4-demethylwyosine (imG-14). << Less

  Mol. Biol. Evol. 27:2062-2077(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Isolation and characterization of the human tRNA-(N1G37) methyltransferase (TRM5) and comparison to the Escherichia coli TrmD protein.

  Brule H., Elliott M., Redlak M., Zehner Z.E., Holmes W.M.

  A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced. The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine. The effects of RNA sequence and structure on the methylation reaction ... >> More

  A human TRM5 cDNA has been cloned and recombinant tRNA-N(1)G37 methyltransferase was produced. The recombinant enzyme methylates the N1 position of guanosine 37 (G37) in selected tRNA transcripts utilizing S-adenosyl methionine. The effects of RNA sequence and structure on the methylation reaction in comparison between the Escherichia coli TrmD and human TRM5 recombinant enzymes are presented. G37-methylation by TRM5 occurs regardless of the nature of the nucleotide at position 36. TRM5 also methylates inosine at position 37 unlike TrmD, which recognizes the G36pG37 motif preferentially and does not methylate inosine. New evidence is presented concerning TrmD showing that with some tRNA species, A at position 36 is also recognized. The TRM5 enzyme is sensitive to subtle changes in the tRNA-protein tertiary interaction leading to loss of activity. The TrmD enzyme is more tolerant of alterations in tRNA-protein tertiary interactions as long as the core tRNA structure and the G36pG37 are present. The TRM5 enzyme does not have an absolute requirement for magnesium ions, whereas TrmD requires magnesium to express activity. TRM5 demonstrates much higher affinity for substrates with K(m) values for tRNA that are nanomolar. TrmD has K(m) values for tRNA in the micromolar range. Recombinant TRM5 appears to function as a 60 772 Da monomer, while recombinant TrmD functions as a homodimer of 30 586 Da subunits. Bioinformatic analysis of the human TRM5 genomic locus (KIAA1393) have identified TRM5 homologues in eukaryotes and archaea; however, no significantly homologous regions were identified in any prokaryotes including the TrmD gene. << Less

  Biochemistry 43:9243-9255(2004) [PubMed] [EuropePMC]

• Yeast mitochondrial initiator tRNA is methylated at guanosine 37 by the Trm5-encoded tRNA (guanine-N1-)-methyltransferase.

  Lee C., Kramer G., Graham D.E., Appling D.R.

  The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes ... >> More

  The TRM5 gene encodes a tRNA (guanine-N1-)-methyltransferase (Trm5p) that methylates guanosine at position 37 (m(1)G37) in cytoplasmic tRNAs in Saccharomyces cerevisiae. Here we show that Trm5p is also responsible for m(1)G37 methylation of mitochondrial tRNAs. The TRM5 open reading frame encodes 499 amino acids containing four potential initiator codons within the first 48 codons. Full-length Trm5p, purified as a fusion protein with maltose-binding protein, exhibited robust methyltransferase activity with tRNA isolated from a Delta trm5 mutant strain, as well as with a synthetic mitochondrial initiator tRNA (tRNA(Met)(f)). Primer extension demonstrated that the site of methylation was guanosine 37 in both mitochondrial tRNA(Met)(f) and tRNA(Phe). High pressure liquid chromatography analysis showed the methylated product to be m(1)G. Subcellular fractionation and immunoblotting of a strain expressing a green fluorescent protein-tagged version of the TRM5 gene revealed that the enzyme was localized to both cytoplasm and mitochondria. The slightly larger mitochondrial form was protected from protease digestion, indicating a matrix localization. Analysis of N-terminal truncation mutants revealed that a Trm5p active in the cytoplasm could be obtained with a construct lacking amino acids 1-33 (Delta1-33), whereas production of a Trm5p active in the mitochondria required these first 33 amino acids. Yeast expressing the Delta1-33 construct exhibited a significantly lower rate of oxygen consumption, indicating that efficiency or accuracy of mitochondrial protein synthesis is decreased in cells lacking m(1)G37 methylation of mitochondrial tRNAs. These data suggest that this tRNA modification plays an important role in reading frame maintenance in mitochondrial protein synthesis. << Less

  J. Biol. Chem. 282:27744-27753(2007) [PubMed] [EuropePMC]

• Crystal structure of archaeal tRNA(m(1)G37)methyltransferase aTrm5.

  Goto-Ito S., Ito T., Ishii R., Muto Y., Bessho Y., Yokoyama S.

  Methylation of the N1 atom of guanosine at position 37 in tRNA, the position 3'-adjacent to the anticodon, generates the modified nucleoside m(1)G37. In archaea and eukaryotes, m(1)G37 synthesis is catalyzed by tRNA(m(1)G37)methyltransferase (archaeal or eukaryotic Trm5, a/eTrm5). Here we report t ... >> More

  Methylation of the N1 atom of guanosine at position 37 in tRNA, the position 3'-adjacent to the anticodon, generates the modified nucleoside m(1)G37. In archaea and eukaryotes, m(1)G37 synthesis is catalyzed by tRNA(m(1)G37)methyltransferase (archaeal or eukaryotic Trm5, a/eTrm5). Here we report the crystal structure of archaeal Trm5 (aTrm5) from Methanocaldococcus jannaschii (formerly known as Methanococcus jannaschii) in complex with the methyl donor analogue at 2.2 A resolution. The crystal structure revealed that the entire protein is composed of three structural domains, D1, D2, and D3. In the a/eTrm5 primary structures, D2 and D3 are highly conserved, while D1 is not conserved. The D3 structure is the Rossmann fold, which is the hallmark of the canonical class-I methyltransferases. The a/eTrm5-defining domain, D2, exhibits structural similarity to some class-I methyltransferases. In contrast, a DALI search with the D1 structure yielded no structural homologues. In the crystal structure, D3 contacts both D1 and D2. The residues involved in the D1:D3 interactions are not conserved, while those participating in the D2:D3 interactions are well conserved. D1 and D2 do not contact each other, and the linker between them is disordered. aTrm5 fragments corresponding to the D1 and D2-D3 regions were prepared in a soluble form. The NMR analysis of the D1 fragment revealed that D1 is well folded by itself, and it did not interact with either the D2-D3 fragment or the tRNA. The NMR analysis of the D2-D3 fragment revealed that it is well folded, independently of D1, and that it interacts with tRNA. Furthermore, the D2-D3 fragment was as active as the full-length enzyme for tRNA methylation. The positive charges on the surface of D2-D3 may be involved in tRNA binding. Therefore, these findings suggest that the interaction between D1 and D3 is not persistent, and that the D2-D3 region plays the major role in tRNA methylation. << Less

  Proteins 72:1274-1289(2008) [PubMed] [EuropePMC]

• Evolution of tRNAPhe:imG2 methyltransferases involved in the biosynthesis of wyosine derivatives in Archaea.

  Urbonavicius J., Rutkiene R., Lopato A., Tauraite D., Stankeviciute J., Aucynaite A., Kaliniene L., van Tilbeurgh H., Meskys R.

  Tricyclic wyosine derivatives are found at position 37 of eukaryotic and archaeal tRNA^Phe In Archaea, the intermediate imG-14 is targeted by three different enzymes that catalyze the formation of yW-86, imG, and imG2. We have suggested previously that a peculiar methyltransferase (aTrm5 ... >> More

  Tricyclic wyosine derivatives are found at position 37 of eukaryotic and archaeal tRNA^Phe In Archaea, the intermediate imG-14 is targeted by three different enzymes that catalyze the formation of yW-86, imG, and imG2. We have suggested previously that a peculiar methyltransferase (aTrm5a/Taw22) likely catalyzes two distinct reactions: N¹-methylation of guanosine to yield m¹G; and C⁷-methylation of imG-14 to yield imG2. Here we show that the recombinant aTrm5a/Taw22-like enzymes from both Pyrococcus abyssi and Nanoarchaeum equitans indeed possess such dual specificity. We also show that substitutions of individual conservative amino acids of P. abyssi Taw22 (P260N, E173A, and R174A) have a differential effect on the formation of m¹G/imG2, while replacement of R134, F165, E213, and P262 with alanine abolishes the formation of both derivatives of G37. We further demonstrate that aTrm5a-type enzyme SSO2439 from Sulfolobus solfataricus, which has no N¹-methyltransferase activity, exhibits C⁷-methyltransferase activity, thereby producing imG2 from imG-14. We thus suggest renaming such aTrm5a methyltransferases as Taw21 to distinguish between monofunctional and bifunctional aTrm5a enzymes. << Less

  RNA 22:1871-1883(2016) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus.

  Takeda H., Toyooka T., Ikeuchi Y., Yokobori S., Okadome K., Takano F., Oshima T., Suzuki T., Endo Y., Hori H.

  Transfer RNA (m(1)G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N(1) atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD rec ... >> More

  Transfer RNA (m(1)G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N(1) atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNA(Phe) transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNA(Phe) harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins. << Less

  Genes Cells 11:1353-1365(2006) [PubMed] [EuropePMC]