tRNA m1A58 Methyltransferase Research Articles

The Arabidopsis TRM61/TRM6 complex is a bona fide tRNA N1-methyladenosine methyltransferase.

tRNA molecules, which contain the most abundant post-transcriptional modifications, are crucial for proper gene expression and protein biosynthesis. Methylation at N1 of adenosine 58 (A58) is critical for maintaining the stability of initiator methionyl-tRNA (tRNAiMet) in bacterial, archaeal, and eukaryotic tRNAs. However, although research has been conducted in yeast and mammals, it remains unclear how A58 in plant tRNAs is modified and involved in development. In this study, we identify the nucleus-localized complex AtTRM61/AtTRM6 in Arabidopsis as tRNA m1A58 methyltransferase. Deficiency or a lack of either AtTRM61 or AtTRM6 leads to embryo arrest and seed abortion. The tRNA m1A level decreases in conditionally complemented Attrm61/LEC1pro::AtTRM61 plants and this is accompanied by reduced levels of tRNAiMet, indicating the importance of the tRNA m1A modification for tRNAiMet stability. Taken together, our results demonstrate that tRNA m1A58 modification is necessary for tRNAiMet stability and is required for embryo development in Arabidopsis.

Crystal Structure of the Human tRNA m1A58 Methyltransferase–tRNA3Lys Complex: Refolding of Substrate tRNA Allows Access to the Methylation Target

Human tRNA3Lys is the primer for reverse transcription of HIV; the 3′ end is complementary to the primer-binding site on HIV RNA. The complementarity ends at the 18th base, A58, which in tRNA3Lys is modified to remove Watson–Crick pairing. Motivated to test the role of the modification in terminating the primer-binding sequence and thus limiting run-on transcription, we asked how the modification of RNA could be accomplished. tRNA m1A58 methyltransferase (m1A58 MTase) methylates N1 of A58, which is buried in the TΨC-loop of tRNA, from cofactor S-adenosyl-l-methionine. This conserved tRNA modification is essential for stability of initiator tRNA in Saccharomyces cerevisiae. Reported here, three structures of human tRNA m1A58 MTase in complex with human tRNA3Lys and the product S-adenosyl-l-homocysteine show a dimer of heterodimers in which each heterodimer comprises a catalytic chain, Trm61, and a homologous but noncatalytic chain, Trm6, repurposed as a tRNA-binding subunit that acts in trans; tRNAs bind across the dimer interface such that Trm6 from the opposing heterodimer brings A58 into the active site of Trm61. T-loop and D-loop are splayed apart showing how A58, normally buried in tRNA, becomes accessible for modification. This result has broad impact on our understanding of the mechanisms of modifying internal sites in folded tRNA. The structures serve as templates for design of inhibitors that could be used to test tRNA m1A58 MTase's impact on retroviral priming and transcription.

Crystal structure of tRNA m(1)A58 methyltransferase TrmI from Aquifex aeolicus in complex with S-adenosyl-L-methionine.

The N 1-methyladenosine residue at position 58 of tRNA is found in the three domains of life, and contributes to the stability of the three-dimensional L-shaped tRNA structure. In thermophilic bacteria, this modification is important for thermal adaptation, and is catalyzed by the tRNA m1A58 methyltransferase TrmI, using S-adenosyl-l-methionine (AdoMet) as the methyl donor. We present the 2.2 Å crystal structure of TrmI from the extremely thermophilic bacterium Aquifex aeolicus, in complex with AdoMet. There are four molecules per asymmetric unit, and they form a tetramer. Based on a comparison of the AdoMet binding mode of A. aeolicus TrmI to those of the Thermus thermophilus and Pyrococcus abyssi TrmIs, we discuss their similarities and differences. Although the binding modes to the N6 amino group of the adenine moiety of AdoMet are similar, using the side chains of acidic residues as well as hydrogen bonds, the positions of the amino acid residues involved in binding are diverse among the TrmIs from A. aeolicus, T. thermophilus, and P. abyssi.

Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs

In human mitochondria, 1-methyladenosine (m¹A) occurs at position 58 of tRNA(Leu(UUR)). In addition, partial m¹A58 modifications have been found in human mitochondrial tRNA(Lys) and tRNA(Ser(UCN)). We identified human Trmt61B, which encodes a mitochondria-specific tRNA methyltransferase responsible for m¹A58 in these three tRNAs. Trmt61B is dominantly localized to the mitochondria. m¹A58 formation in human mitochondrial tRNA(Leu(UUR)) could be reconstituted in vitro using recombinant Trmt61B in the presence of Ado-Met as a methyl donor. Unlike the cytoplasmic tRNA m¹A58 methyltransferase that consists of an α2β2 heterotetramer formed by Trmt61A and Trmt6, Trmt61B formed a homo-oligomer (presumably a homotetramer) that resembled the bacterial homotetrameric m¹A58 methyltransferase. The bacterial origin of Trmt61B is supported by the results of the phylogenetic analysis.

Crystal Structure of Thermus thermophilus tRNA m1A58 Methyltransferase and Biophysical Characterization of Its Interaction with tRNA

Methyltransferases from the m1A58 tRNA methyltransferase (TrmI) family catalyze the S-adenosyl-l-methionine-dependent N1-methylation of tRNA adenosine 58. The crystal structure of Thermus thermophilus TrmI, in complex with S-adenosyl-l-homocysteine, was determined at 1.7 Å resolution. This structure is closely related to that of Mycobacterium tuberculosis TrmI, and their comparison enabled us to enlighten two grooves in the TrmI structure that are large enough and electrostatically compatible to accommodate one tRNA per face of TrmI tetramer. We have then conducted a biophysical study based on electrospray ionization mass spectrometry, site-directed mutagenesis, and molecular docking. First, we confirmed the tetrameric oligomerization state of TrmI, and we showed that this protein remains tetrameric upon tRNA binding, with formation of complexes involving one to two molecules of tRNA per TrmI tetramer. Second, three key residues for the methylation reaction were identified: the universally conserved D170 and two conserved aromatic residues Y78 and Y194. We then used molecular docking to position a N9-methyladenine in the active site of TrmI. The N9-methyladenine snugly fits into the catalytic cleft, where the side chain of D170 acts as a bidentate ligand binding the amino moiety of S-adenosyl-l-methionine and the exocyclic amino group of the adenosine. Y194 interacts with the N9-methyladenine ring, whereas Y78 can stabilize the sugar ring. From our results, we propose that the conserved residues that form the catalytic cavity (D170, Y78, and Y194) are essential for fashioning an optimized shape of the catalytic pocket.

Nuclear surveillance and degradation of hypomodified initiator tRNAMet in S. cerevisiae.

The tRNA m(1)A58 methyltransferase is composed of two subunits encoded by the essential genes TRM6 and TRM61 (formerly GCD10 and GCD14). The trm6-504 mutation results in a defective m(1)A methyltransferase (Mtase) and a temperature-sensitive growth phenotype that is attributable to the absence of m(1)A58 and consequential tRNA(i)(Met) instability. We used a genetic approach to identify the genes responsible for tRNA(i)(Met) degradation in trm6 cells. Three recessive extragenic mutations that suppress trm6-504 mutant phenotypes and restore hypomodified tRNA(i)(Met) to near normal levels were identified. The wild-type allele of one suppressor, DIS3/RRP44, encodes a 3'-5' exoribonuclease and a member of the multisubunit exosome complex. We provide evidence that a functional nuclear exosome is required for the degradation of tRNA(i)(Met) lacking m(1)A58. A second suppressor gene encodes Trf4p, a DNA polymerase (pol sigma) with poly(A) polymerase activity. Whereas deletion of TRF4 leads to stabilization of tRNA(i)(Met), overexpression of Trf4p destabilizes the hypomodified tRNA(i)(Met) in trm6 cells. The hypomodified, but not wild-type, pre-tRNA(i)(Met) accumulates as a polyadenylated species, whose abundance and length distribution both increase upon Trf4p overexpression. These data indicate that a tRNA surveillance pathway exists in yeast that requires Trf4p and the exosome for polyadenylation and degradation of hypomodified pre-tRNA(i)(Met).