N1-methyladenosine (m1A) is found at position 58 in the T-loop of many tRNAs. In yeast, the formation of this modified nucleoside is catalyzed by the essential tRNA (m1A58) methyltransferase, a tetrameric enzyme that is composed of two types of subunits (Gcd14p and Gcd10p). In this report we describe the cloning, expression and characterization of a Gcd14p homolog from the hyperthermophilic bacterium Thermus thermophilus. The purified recombinant enzyme behaves as a homotetramer of ∼150 kDa by gel filtration and catalyzes the site- specific formation of m1A at position 58 of the T-loop of tRNA in the absence of any other complementary protein. S-adenosylmethionine is used as donor of the methyl group. Thus, we propose to name the bacterial enzyme TrmI and accordingly its structural gene trmI. These results provide a key evolutionary link between the functionally characterized two-component eukaryotic enzyme and the recently described crystal structure of an uncharacterized, putative homotetrameric methyltransferase Rv2118c from Mycobacterium tuberculosis. Interest ingly, inactivation of the T.thermophilus trmI gene results in a thermosensitive phenotype (growth defect at 80°C), which suggests a role of the N1-methylation of tRNA adenosine-58 in adaptation of life to extreme temperatures.
Methyltransferases (MTases) form a major class of tRNA-modifying enzymes needed for the proper functioning of tRNA. Recently, RNA MTases from the TrmN/Trm14 family that are present in Archaea, Bacteria and Eukaryota have been shown to specifically modify tRNAPhe at guanosine 6 in the tRNA acceptor stem. Here, we report the first X-ray crystal structures of the tRNA m2G6 (N2-methylguanosine) MTase TTCTrmN from Thermus thermophilus and its ortholog PfTrm14 from Pyrococcus furiosus. Structures of PfTrm14 were solved in complex with the methyl donor S-adenosyl-l-methionine (SAM or AdoMet), as well as the reaction product S-adenosyl-homocysteine (SAH or AdoHcy) and the inhibitor sinefungin. TTCTrmN and PfTrm14 consist of an N-terminal THUMP domain fused to a catalytic Rossmann-fold MTase (RFM) domain. These results represent the first crystallographic structure analysis of proteins containing both THUMP and RFM domain, and hence provide further insight in the contribution of the THUMP domain in tRNA recognition and catalysis. Electrostatics and conservation calculations suggest a main tRNA binding surface in a groove between the THUMP domain and the MTase domain. This is further supported by a docking model of TrmN in complex with tRNAPhe of T. thermophilus and via site-directed mutagenesis.
In Saccharomyces cerevisiae, a two-subunit methyltransferase (Mtase) encoded by the essential genes TRM6 and TRM61 is responsible for the formation of 1-methyladenosine, a modified nucleoside found at position 58 in tRNA that is critical for the stability of tRNAiMet. The crystal structure of the homotetrameric m1A58 tRNA Mtase from Mycobacterium tuberculosis, TrmI, has been solved and was used as a template to build a model of the yeast m1A58 tRNA Mtase heterotetramer. We altered amino acids in TRM6 and TRM61 that were predicted to be important for the stability of the heteroligomer based on this model. Yeast strains expressing trm6 and trm61 mutants exhibited growth phenotypes indicative of reduced m1A formation. In addition, recombinant mutant enzymes had reduced in vitro Mtase activity. We demonstrate that the mutations introduced do not prevent heteroligomer formation and do not disrupt binding of the cofactor S-adenosyl-l-methionine. Instead, amino acid substitutions in either Trm6p or Trm61p destroy the ability of the yeast m1A58 tRNA Mtase to bind tRNAiMet, indicating that each subunit contributes to tRNA binding and suggesting a structural alteration of the substrate-binding pocket occurs when these mutations are present.
Two archaeal tRNA methyltransferases belonging to the SPOUT superfamily and displaying unexpected activities are identified. These enzymes are orthologous to the yeast Trm10p methyltransferase, which catalyses the formation of 1-methylguanosine at position 9 of tRNA. In contrast, the Trm10p orthologue from the crenarchaeon Sulfolobus acidocaldarius forms 1-methyladenosine at the same position. Even more surprisingly, the Trm10p orthologue from the euryarchaeon Thermococcus kodakaraensis methylates the N1-atom of either adenosine or guanosine at position 9 in different tRNAs. This is to our knowledge the first example of a tRNA methyltransferase with a broadened nucleoside recognition capability. The evolution of tRNA methyltransferases methylating the N1 atom of a purine residue is discussed.
The S-adenosyl-l-methionine dependent methylation of adenine 58 in the T-loop of tRNAs is essential for cell growth in yeast or for adaptation to high temperatures in thermophilic organisms. In contrast to bacterial and eukaryotic tRNA m1A58 methyltransferases that are site-specific, the homologous archaeal enzyme from Pyrococcus abyssi catalyzes the formation of m1A also at the adjacent position 57, m1A57 being a precursor of 1-methylinosine. We report here the crystal structure of P. abyssi tRNA m1A57/58 methyltransferase (PabTrmI), in complex with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine in three different space groups. The fold of the monomer and the tetrameric architecture are similar to those of the bacterial enzymes. However, the inter-monomer contacts exhibit unique features. In particular, four disulfide bonds contribute to the hyperthermostability of the archaeal enzyme since their mutation lowers the melting temperature by 16.5°C. His78 in conserved motif X, which is present only in TrmIs from the Thermococcocales order, lies near the active site and displays two alternative conformations. Mutagenesis indicates His78 is important for catalytic efficiency of PabTrmI. When A59 is absent in tRNAAsp, only A57 is modified. Identification of the methylated positions in tRNAAsp by mass spectrometry confirms that PabTrmI methylates the first adenine of an AA sequence.
tRNA m1A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.
In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.
The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.
N7-methylguanine at position 46 (m7G46) in tRNA is produced by tRNA (m7G46) 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 m7G46 modification in tRNAPhe 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 m7G46 modification. Assays with tRNA modification enzymes revealed hypo-modifications of Gm18 and m1G37, suggesting that the m7G46 positively affects their formations. Although the lack of the m7G46 modification and the hypo-modifications do not affect the Phe charging activity of tRNAPhe, they cause a decrease in melting temperature of class I tRNA and degradation of tRNAPhe and tRNAIle. 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 m7G46 modification is required for cell viability at high temperatures via a tRNA modification network, in which the m7G46 modification supports introduction of other modifications.
Methyltransferases that use S-adenosylmethionine (AdoMet) as a cofactor to catalyse 5-methyl uridine (m5U) formation in tRNAs and rRNAs are widespread in Bacteria and Eukaryota, and are also found in certain Archaea. These enzymes belong to the COG2265 cluster, and the Gram-negative bacterium Escherichia coli possesses three paralogues. These comprise the methyltransferases TrmA that targets U54 in tRNAs, RlmC that modifies U747 in 23S rRNA and RlmD that is specific for U1939 in 23S rRNA. The tRNAs and rRNAs of the Gram-positive bacterium Bacillus subtilis have the same three m5U modifications. However, as previously shown, the m5U54 modification in B. subtilis tRNAs is catalysed in a fundamentally different manner by the folate-dependent enzyme TrmFO, which is unrelated to the E. coli TrmA. Here, we show that methylation of U747 and U1939 in B. subtilis rRNA is catalysed by a single enzyme, YefA that is a COG2265 member. A recombinant version of YefA functions in an E. coli m5U-null mutant adding the same two rRNA methylations. The findings suggest that during evolution, COG2265 enzymes have undergone a series of changes in target specificity and that YefA is closer to an archetypical m5U methyltransferase. To reflect its dual specificity, YefA is renamed RlmCD.
Modified nucleosides in tRNAs play important roles in tRNA structure, biosynthesis and function, and serve as crucial determinants of bacterial growth and virulence. In the yeast Saccharomyces cerevisiae, mutants defective in N1-methylation of a highly conserved adenosine (A58) in the TΨC loop of initiator tRNA are non-viable. The yeast m1A58 methyltransferase is a heterotetramer consisting of two different polypeptide chains, Gcd14p and Gcd10p. Interestingly, while m1A58 is not found in most eubacteria, the mycobacterial tRNAs have m1A58. Here, we report on the cloning, overexpression, purification and biochemical characterization of the Rv2118c gene-encoded protein (Rv2118p) from Mycobacterium tuberculosis, which is homologous to yeast Gcd14p. We show that Rv2118c codes for a protein of ∼31 kDa. Activity assays, modified base analysis and primer extension experiments using reverse transcriptase reveal that Rv2118p is an S-adenosyl-l-methionine-dependent methyltransferase which carries out m1A58 modification in tRNAs, both in vivo and in vitro. Remarkably, when expressed in Escherichia coli, the enzyme methylates the endogenous E.coli initiator tRNA essentially quantitatively. Furthermore, unlike its eukaryotic counterpart, which is a heterotetramer, the mycobacterial enzyme is a homotetramer. Also, the presence of rT modification at position 54, which was found to inhibit the Tetrahymena pyriformis enzyme, does not affect the activity of Rv2118p. Thus, the mycobacterial m1A58 tRNA methyltransferase possesses distinct biochemical properties. We discuss aspects of the biological relevance of Rv2118p in M.tuberculosis, and its potential use as a drug target to control the growth of mycobacteria.
The 5-methyluridine is invariably found at position 54 in the TΨC loop of tRNAs of most organisms. In Pyrococcus abyssi, its formation is catalyzed by the S-adenosyl-l-methionine-dependent tRNA (uracil-54, C5)-methyltransferase (PabTrmU54), an enzyme that emerged through an ancient horizontal transfer of an RNA (uracil, C5)-methyltransferase-like gene from bacteria to archaea. The crystal structure of PabTrmU54 in complex with S-adenosyl-l-homocysteine at 1.9 Å resolution shows the protein organized into three domains like Escherichia coli RumA, which catalyzes the same reaction at position 1939 of 23S rRNA. A positively charged groove at the interface between the three domains probably locates part of the tRNA-binding site of PabTrmU54. We show that a mini-tRNA lacking both the D and anticodon stem-loops is recognized by PabTrmU54. These results were used to model yeast tRNAAsp in the PabTrmU54 structure to get further insights into the different RNA specificities of RumA and PabTrmU54. Interestingly, the presence of two flexible loops in the central domain, unique to PabTrmU54, may explain the different substrate selectivities of both enzymes. We also predict that a large TΨC loop conformational change has to occur for the flipping of the target uridine into the PabTrmU54 active site during catalysis.
Transfer RNA (tRNA) structure, modifications and functions are evolutionary and established in bacteria, archaea and eukaryotes.
Typically the tRNA modifications are indispensable for its stability and are required for decoding the mRNA into amino acids for
protein synthesis. A conserved methylation has been located on the anticodon loop specifically at the 37th position and it is next to
the anticodon bases. This modification is called as m1G37 and it is catalyzed by tRNA (m1G37) methyltransferase (TrmD). It is
deciphered that G37 positions occur on few additional amino acids specific tRNA subsets in bacteria. Furthermore, Archaea and
Eukaryotes have more number of tRNA subsets which contains G37 position next to the anticodon and the G residue are located at
different positions such as G36, G37, G38, 39, and G40. In eight bacterial species, G (guanosine) residues are presents at the 37th and
38th position except three tRNA subsets having G residues at 36th and 39th positions. Therefore we propose that m1G37 modification
may be feasible at 36th, 37th, 38th, 39th and 40th positions next to the anticodon of tRNAs. Collectively, methylation at G residues
close to the anticodon may be possible at different positions and without restriction of anticodon 3rd base A, C, U or G.
tRNAs; Codon; Anticodon; m1G modification
The Escherichia coli trmA gene encodes the tRNA(m5U54)methyltransferase, which catalyses the formation of m5U54 in tRNA. During the synthesis of m5U54, a covalent 62-kDa TrmA-tRNA intermediate is formed between the amino acid C324 of the enzyme and the 6-carbon of uracil. We have analysed the formation of this TrmA-tRNA intermediate and m5U54 in vivo, using mutants with altered TrmA. We show that the amino acids F188, Q190, G220, D299, R302, C324 and E358, conserved in the C-terminal catalytic domain of several RNA(m5U)methyltransferases of the COG2265 family, are important for the formation of the TrmA-tRNA intermediate and/or the enzymatic activity. These amino acids seem to have the same function as the ones present in the catalytic domain of RumA, whose structure is known, and which catalyses the formation of m5U in position 1939 of E. coli 23 S rRNA. We propose that the unusually high in vivo level of the TrmA-tRNA intermediate in wild-type cells may be due to a suboptimal cellular concentration of SAM, which is required to resolve this intermediate. Our results are consistent with the modular evolution of RNA(m5U)methyltransferases, in which the specificity of the enzymatic reaction is achieved by combining the conserved catalytic domain with different RNA-binding domains.
tRNA (adenine-1) methyltransferase occurs in Bacillus subtilis. Eucaryotic tRNAThr and tRNATyr from yeast in which 1-methyladenosine (m1A) is already present in the TpsiC loop, can be methylated in vitro with S-adenosylmethionine and B. subtilis extracts. Each of the specific tRNAs accepts 1 mol of methyl groups per mol tRNA. The enzyme transforms into m1A the 3'-terminal adenylic acid residue of the dihydrouridine loop, a new position for a modified adenosine residue in tRNA. Both tRNAs have the sequence Py-A-A-G-G-C-m2(2)G in the D-loop and D-stem region. Other tRNAs with the same sequence in this region also serve as substrates for the tRNA (adenine-1) methyltransferase.
The expression, purification, crystallization and X-ray diffraction analysis of tRNA m1A58 methyltransferase from S. cerevisiae are reported.
In Saccharomyces cerevisiae, TRM6 and TRM61 compose a tRNA methyltransferase which catalyzes the methylation of the N1 of adenine at position 58 in tRNAs, especially initiator methionine tRNA. TRM61 is the subunit that binds S-adenosyl-l-methionine and both subunits contribute to target tRNA binding. In order to elucidate the catalytic mechanism of TRM6–TRM61 and the mode of interaction between the two subunits, expression, purification, crystallization and X-ray diffraction analysis of the TRM6–TRM61 complex were performed in this study. The crystals diffracted to 2.80 Å resolution and belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 139.14, c = 101.62 Å.
tRNA m1A58 methyltransferase; Saccharomyces cerevisiae
N1-methylation of adenosine to m1A occurs in several different positions in tRNAs from various organisms. A methyl group at position N1 prevents Watson–Crick-type base pairing by adenosine and is therefore important for regulation of structure and stability of tRNA molecules. Thus far, only one family of genes encoding enzymes responsible for m1A methylation at position 58 has been identified, while other m1A methyltransferases (MTases) remain elusive. Here, we show that Bacillus subtilis open reading frame yqfN is necessary and sufficient for N1-adenosine methylation at position 22 of bacterial tRNA. Thus, we propose to rename YqfN as TrmK, according to the traditional nomenclature for bacterial tRNA MTases, or TrMet(m1A22) according to the nomenclature from the MODOMICS database of RNA modification enzymes. tRNAs purified from a ΔtrmK strain are a good substrate in vitro for the recombinant TrmK protein, which is sufficient for m1A methylation at position 22 as are tRNAs from Escherichia coli, which natively lacks m1A22. TrmK is conserved in Gram-positive bacteria and present in some Gram-negative bacteria, but its orthologs are apparently absent from archaea and eukaryota. Protein structure prediction indicates that the active site of TrmK does not resemble the active site of the m1A58 MTase TrmI, suggesting that these two enzymatic activities evolved independently.
tRNA (m7G46) methyltransferase from E. coli was overexpressed, purified and crystallized. Diffraction data were collected to 2.04 Å resolution.
Transfer RNA (tRNA) (m7G46) methyltransferase (TrmB) belongs to the Rossmann-fold methyltransferase (RFM) family and uses S-adenosyl-l-methionine (SAM) as the methyl-group donor to catalyze the formation of N
7-methylguanosine (m7G) at position 46 in the variable loop of tRNAs. After attempts to crystallize full-length Escherichia coli TrmB (EcTrmB) failed, a truncated protein lacking the first 32 residues of the N-terminus but with an additional His6 tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 3350 (PEG 3350) as precipitant at 283 K. An X-ray diffraction data set was collected using a single flash-cooled crystal that belonged to space group P21.
tRNA (m7G46) methyltransferase; Rossmann-fold methyltransferase family
Unlike other transfer RNAs (tRNA)-modifying enzymes from the SPOUT methyltransferase superfamily, the tRNA (Um34/Cm34) methyltransferase TrmL lacks the usual extension domain for tRNA binding and consists only of a SPOUT domain. Both the catalytic and tRNA recognition mechanisms of this enzyme remain elusive. By using tRNAs purified from an Escherichia coli strain with the TrmL gene deleted, we found that TrmL can independently catalyze the methyl transfer from S-adenosyl-L-methionine to and isoacceptors without the involvement of other tRNA-binding proteins. We have solved the crystal structures of TrmL in apo form and in complex with S-adenosyl-homocysteine and identified the cofactor binding site and a possible active site. Methyltransferase activity and tRNA-binding affinity of TrmL mutants were measured to identify residues important for tRNA binding of TrmL. Our results suggest that TrmL functions as a homodimer by using the conserved C-terminal half of the SPOUT domain for catalysis, whereas residues from the less-conserved N-terminal half of the other subunit participate in tRNA recognition.
Guanosine at position 26 in eukaryotic tRNAs is usually modified to N2 , N2 -dimethylguanosine (m22G26). In Saccharomyces cerevisiae , this reaction is catalysed by the TRM1 encoded tRNA (m22G26)dimethyltransferase. As a prerequisite for future studies, the yeast TRM1 gene was expressed in Escherichia coli and the His-tagged Trm1 protein (rTrm1p) was extensively purified. rTrm1p catalysed both the mono- and dimethylation of G26 in vivo in Escherichia coli tRNA and in vitro in yeast trm1 mutant tRNA. The TRM1 gene from two independent wild-type yeast strains differed at 14 base positions causing two amino acid exchanges . Exchange of the original Ser467 for Leu caused a complete loss of enzyme activity in vitro against trm1 yeast tRNA. Comparatively short N- or C-terminal deletions from the 570 amino acid long Trm1 polypeptide decreased or eliminated the enzyme activity, as did some point mutations within these regions. This indicated that the protein is not a two domain peptide with the enzyme activity localised to one of the domains, but rather that both ends of the polypeptide seem to interact to influence the conformation of those parts that make up the RNA-binding site and/or the active site of the enzyme.
The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.
Using a microarray that tiles all known yeast non-coding RNAs, we compared RNA from wild-type cells with RNA from mutants encoding known and putative RNA modifying enzymes. We show that at least five types of RNA modification (dihydrouridine, m1G, m22G, m1A and m26A) catalyzed by 10 different enzymes (Trm1p, Trm5, Trm10p, Dus1p-Dus4p, Dim1p, Gcd10p and Gcd14p) can be detected by virtue of differential hybridization to oligonucleotides on the array that are complementary to the modified sites. Using this approach, we identified a previously undetected m1A modification in GlnCTG tRNA, the formation of which is catalyzed by the Gcd10/Gcd14 complex.
Transfer RNAs of the extreme halophile Haloferax volcanii contain several modified nucleosides, among them 1-methylpseudouridine (m1 psi), pseudouridine (psi), 2'-0-methylcytosine (Cm) and 1-methylinosine (m1l), present in positions 54, 55, 56 and 57 of the psi-loop, respectively. At the same positions in tRNAs from eubacteria and eukaryotes, ribothymidine (T-54), pseudouridine (psi-55), non-modified cytosine (C-56) and non-modified adenosine or guanosine (A-57 or G-57) are found in the so-called T psi-loop. Using as substrate a T7 transcript of Haloferax volcanii tRNA(Ile) devoid of modified nucleosides, the enzymatic activities of several tRNA modification enzymes, including those for m1 psi-54, psi-55, Cm-56 and m1l-57, were detected in cell extracts of H.volcanii. Here, we demonstrate that modification of A-57 into m1l-57 in H.volcanii tRNA(Ile) occurs via a two-step enzymatic process. The first step corresponds to the formation of m1A-57 catalyzed by a S-adenosylmethionine-dependent tRNA methyltransferase, followed by the deamination of the 6-amino group of the adenine moiety by a 1-methyladenosine-57 deaminase. This enzymatic pathway differs from that leading to the formation of m1l-37 in the anticodon loop of eukaryotic tRNA(Ala). In the latter case, inosine-37 formation preceeds the S-adenosylmethionine-dependent methylation of l-37 into m1l-37. Thus, enzymatic strategies for catalyzing the formation of 1-methylinosine in tRNAs differ in organisms from distinct evolutionary kingdoms.
Throughout the kingdoms of life, transfer RNA (tRNA) undergoes over 100 enzyme-catalyzed, methyl-based modifications. Although a majority of the methylations are conserved from bacteria to mammals, the functions of a number of these modifications are unknown. Many of the proteins responsible for tRNA methylation, named tRNA methyltransferases (Trms), have been characterized in Saccharomyces cerevisiae. In contrast, only a few human Trms have been characterized. A BLAST search for human homologs of each S. cerevisiae Trm revealed a total of 34 human proteins matching our search criteria for an S. cerevisiae Trm homolog candidate. We have compiled a database cataloging basic information about each human and yeast Trm. Every S. cerevisiae Trm has at least one human homolog, while several Trms have multiple candidates. A search of cancer cell versus normal cell mRNA expression studies submitted to Oncomine found that 30 of the homolog genes display a significant change in mRNA expression levels in at least one data set. While 6 of the 34 human homolog candidates have confirmed tRNA methylation activity, the other candidates remain uncharacterized. We believe that our database will serve as a resource for investigating the role of human Trms in cellular stress signaling.
A tRNA (guanine-2) methyltransferase has been purified to homogeneity from the protozoan Tetrahymena pyriformis. The enzyme methylates purified E. coli tRNAs which have a guanine residue at position 26 from the 5' end; it also methylates tRNA prepared from the m22G- yeast mutant trm 1. This methyltransferase is therefore equivalent to the guanine methyltransferase 2mGII found in mammalian extracts. The purified 2mGII from Tetrahymena is capable of forming both N2-methylguanine and N22-dimethylguanine on a single tRNA isoaccepting species; under conditions of limiting tRNA or long reaction times the predominant product is dimethylguanine. Analysis of the products formed under varying reaction conditions suggests that dimethylguanine formation is a two step process requiring dissociation of the enzyme-monomethylated tRNA intermediate.
Gcd10p and Gcd14p were first identified genetically as repressors of GCN4 mRNA translation in Saccharomyces cerevisiae. Recent findings indicate that Gcd10p and Gcd14p reside in a nuclear complex required for the presence of 1-methyladenosine in tRNAs. Here we show that Gcd14p is an essential protein with predicted binding motifs for S-adenosylmethionine, consistent with a direct function in tRNA methylation. Two different gcd14 mutants exhibit defects in cell growth and accumulate high levels of initiator methionyl-tRNA (tRNAiMet) precursors containing 5′ and 3′ extensions, suggesting a defect in processing of the primary transcript. Dosage suppressors of gcd10 mutations, encoding tRNAiMet (hcIMT1 to hcIMT4; hc indicates that the gene is carried on a high-copy-number plasmid) or a homologue of human La protein implicated in tRNA 3′-end formation (hcLHP1), also suppressed gcd14 mutations. In fact, the lethality of a GCD14 deletion was suppressed by hcIMT4, indicating that the essential function of Gcd14p is required for biogenesis of tRNAiMet. A mutation in GCD10 or deletion of LHP1 exacerbated the defects in cell growth and expression of mature tRNAiMet in gcd14 mutants, consistent with functional interactions between Gcd14p, Gcd10p, and Lhp1p in vivo. Surprisingly, the amounts of NME1 and RPR1, the RNA components of RNases P and MRP, were substantially lower in gcd14 lhp1::LEU2 double mutants than in the corresponding single mutants, whereas 5S rRNA was present at wild-type levels. Our findings suggest that Gcd14p and Lhp1p cooperate in the maturation of a subset of RNA polymerase III transcripts.
Correct codon-anticodon pairing promotes translational fidelity, with these interactions greatly facilitated by modified nucleosides found in tRNA. We hypothesized that wobble uridine modifications catalyzed by tRNA methyltransferase 9 (Trm9) are essential for translational fidelity. In support, we have used phenotypic, reporter and protein-based assays to demonstrate increased translational infidelity in trm9Δ Saccharomyces cerevisiae cells. Codon reengineering studies suggest that Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific genes, those rich in arginine and glutamic acid codons from mixed boxes. Using quantitative tRNA modification analysis, we determined that trm9Δ cells are only deficient in 2 of 23 tRNA modifications, with those 2, 5-methoxycarbonylmethyluridine (mcm5U) and 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U), classified as key determinants of translational fidelity. We also show that in the absence of mcm5U and mcm5s2U, the resulting translational infidelity promotes protein errors and activation of unfolded protein and heat shock responses. These data support a model in which Trm9-catalyzed tRNA modifications promote fidelity during the translation of specific transcripts, with decreased wobble base modification leading to translational infidelity, protein errors and activation of protein stress response pathways.
Trm9; codon; heat shock response; modification; protein stress; tRNA; translation infidelity; unfolded protein response; wobble base