Novel Methyltransferase for Modified Uridine Residues at the Wobble Position of tRNA

Abstract

We have identified a novel tRNA methyltransferase in Saccharomyces cerevisiae that we designate Trm9. This enzyme, the product of theYML014w gene, catalyzes the esterification of modified uridine nucleotides, resulting in the formation of 5-methylcarbonylmethyluridine in tRNA Arg3 and 5-methylcarbonylmethyl-2-thiouridine in tRNA Glu . In intact yeast cells, disruption of the TRM9 gene results in the complete loss of these modified wobble bases and increased sensitivity at 37°C to paromomycin, a translational inhibitor. These results suggest a role for this potentially reversible methyl esterification reaction when cells are under stress.In the last few years, it has become increasingly evident that S-adenosylmethionine (AdoMet)-dependent methyltransferases play a wide range of roles in cell physiology. Unlike other covalent modifications such as phosphorylation that occur mainly on proteins, methylation has been observed in DNA, RNA, proteins, lipids, polysaccharides, and small molecules (15,35,36,38). As a result of such diversity, identifying the methyltransferases and their substrates has been challenging. Although more than 25 methylated nucleotides have been documented for eukaryotic tRNAs alone, only a few of the corresponding enzymes and their genes have been identified (16,47).In 1999, using conserved AdoMet binding motifs, we identified 26 putative methyltransferases (designated F1 to F26) encoded by the genome of the yeast Saccharomyces cerevisiae (40). Biochemically, we have been able to show that two of these species are in fact methyltransferases; F3 (YDR465c) catalyzes the transfer of a methyl group to the delta-nitrogen atom of arginine residues in a novel protein posttranslational reaction (40, 58), while F9 (YER175c) catalyzes the methyl esterification of the small molecule trans-aconitate (9). Recently, F1 (YDL201w) was found to be responsible for formation of 7-methylguanosine at position 46 of tRNA (3).One major approach in identifying novel enzymes has been the comparison of the methylation spectra of strains lacking the putative methyltransferase and those of their isogenic wildtype parents by separation techniques such as sodium dodecyl sulfate (SDS) gel electrophoresis (26, 59). In the yeast S. cerevisiae, one can study biological methylation in vivo by incubating cells with, the major biological methyl donor (26). As a result, all methyl-accepting species such as RNAs, proteins, and small molecules can become radiolabeled and the fate of the methylated species can be followed biochemically. One can then look for differences in the methylation spectra between a mutant strain and its parent.In all domains of life, tRNAs are highly modified posttranscriptionally (46, 47). Methylation reactions account for the majority of these modifications. In the yeast S. cerevisiae, eight tRNA methyltransferases have been identified so far. The TRM1 gene product forms N 2 ,N 2 -dimethylguanosine at position 26 (14). TRM2 encodes a protein that forms 5-methyluridine at position 54 (41). TRM3 encodes a pr...

Elongator, a conserved complex required for wobble uridine modifications in Eukaryotes

“…17 Inactivation of the other S. cerevisiae Elongator genes encoding Elp1-Elp2p and Elp4-Elp6p displayed identical phenotypes as the elp3 null mutant including the tRNA modification defect. 17,18 This observation suggested that the Elongator complex is required for synthesis of the first step, an intermediate likely to be 5-carboxymethyluridine (cm 5 U) [19][20][21][22][23] , in formation of mcm 5 U, mcm 5 s 2 U, ncm 5 U and ncm 5 U m at U 34 in tRNA (Fig. 1).…”

“…2). [20][21][22][23]52,53 Other than being a subunit required for Trm9p activity, Trm112p is also a subunit required for the activity of 3 other methyltransferases, the tRNA methyltransferase Trm11p forming m 2 G 10 in tRNA, the ribosomal methyl transferase Bud23p methylating G1575 in 18S rRNA 54 and Mtq2p methylating the release factor eRF1.…”

Protein folding and tRNA biology

“…In regard to tRNA modifications, their functions include: stability and flexibility of the tRNA structure (Motorin and Helm 2010), translational fidelity via codon-anticodon interaction (Saint-Léger and Ribas de Pouplana 2015), reading frame maintenance (Waas et al 2007;Delaunay et al 2016;Klassen et al 2016a), tRNA discrimination , nonsense suppression (Benko et al 2000), tRNA stability (Alexandrov et al 2006;Kotelawala et al 2008;Dewe et al 2012), proteome integrity (Nedialkova and Leidel 2015), sensitivity to aminoglycoside antibiotics acting at the decoding site of ribosomes (Kalhor and Clarke 2003), response to stress (Kamenski et al 2007;Chan et al 2012;Gu et al 2014), and recognition of tRNA as either self or nonself by TLR7 receptor (Kaiser et al 2014;Rimbach et al 2015). For general reviews, see Helm (2011), Hopper (2013) and Phizicky and Hopper (2015).…”

Transfer RNA Post-Transcriptional Processing, Turnover, and Subcellular Dynamics in the YeastSaccharomyces cerevisiae

“…Likewise, the absence of pseudouridine (c) at tRNA Tyr position 35 or m 5 C 34 in tRNA Leu CAA causes defects in tRNA-mediated nonsense suppression (Johnson and Abelson 1983;Strobel and Abelson 1986). Alterations of other modifications at position 34 such as ncm 5 Um 34 result in sensitivity to the aminoglycoside antibiotic paromomycin that causes misreading of near cognate codons (Kalhor and Clarke 2003). Some of the subunits of the enzyme responsible for ncm 5 Um 34 and ncm 5 s 2 U 34 modification were previously identified as components of the elongator complex that functions in transcriptional elongation, silencing at telomeres, and DNA damage response; it since has been shown that the phenotypes are indirect consequences of wobble position errors in translation of proteins that function in these processes (Chen et al 2011).…”