The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2′-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs

Cavaillé, Jérôme; Chetouani, Farid; Bachellerie, Jean‐Pierre

doi:10.1017/s1355838299981475

Cited by 93 publications

(110 citation statements)

References 76 publications

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“…This suggests that there are other determinants for this and other modifications, including dihydrouri- dine modification in other organisms. Interestingly, mutations in tRNA Ser (G18C and G19C, separately) not only abolished Gm 18 modification but also blocked the formation of D 20 in tRNA Ser , suggesting the dependence of the tertiary structure for D 20 modification (28).…”

Section: Discussionmentioning

confidence: 97%

The Specificities of Four Yeast Dihydrouridine Synthases for Cytoplasmic tRNAs

Xing

Hiley

Hughes

et al. 2004

Journal of Biological Chemistry

100

114

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Dihydrouridine is a highly abundant modified nucleoside found widely in tRNAs of eubacteria, eukaryotes, and some archaea. In cytoplasmic tRNA of Saccharomyces cerevisiae, dihydrouridine occurs exclusively at positions 16, 17, 20, 20A, 20B, and 47. Here we show that the known dihydrouridine synthases Dus1p and Dus2p and two previously uncharacterized homologs, Dus3p (encoded by YLR401c) and Dus4p (YLR405w), are required for all of the dihydrouridine modification of cytoplasmic tRNAs in S. cerevisiae. We have mapped the in vivo position specificity of the four Dus proteins, by three complementary approaches: determination of the molar ratio of dihydrouridine in purified tRNAs from different dus mutants; microarray analysis of a large number of tRNAs based on differential hybridization of uridineand dihydrouridine-containing tRNAs to the complementary oligonucleotides; and the development and use of a novel dihydrouridine mapping technique, employing primer extension. We show that each of the four Dus proteins has a distinct position specificity: Dus1p for U 16 and U 17 , Dus2p for U 20 , Dus3p for U 47 , and Dus4p for U 20a and U 20b .A ubiquitous feature of tRNAs is the presence of numerous base and ribose modifications (1). More than 80 different RNA modifications have been described (2), 25 of which are found in cytoplasmic tRNAs of the yeast Saccharomyces cerevisiae; these occur at 35 positions, leading to about 11 modifications in an average yeast tRNA (3).Dihydrouridine is among the most abundant modified nucleosides found in tRNA (3); the 905 dihydrouridines found in the 561 curated tRNAs are second in number only to the 1,234 pseudouridines. Consistent with its abundance, dihydrouridine is found widely in tRNAs of eubacteria and eukaryotes (3), although it is less common in archaebacteria (4). Furthermore, dihydrouridines are found at one or more of multiple different positions in tRNA, the vast majority of which are in the D loop at positions 16, 17, 20, 20a, and 20b and at the base of the variable arm at position 47. Only in six exceptional cases is dihydrouridine found elsewhere, and in these cases it occurs at one of four other positions in the D loop (15, 17a, 19, and 21) and at position 48 in the variable arm. The persistent occurrence of dihydrouridine in these positions in so many different organisms underscores the evolutionary importance of the modification and of the sites of modification.To begin to address the roles of dihydrouridine modifications, an important first step is to decipher the substrate specificity rules for the various modified dihydrouridine residues of tRNA. The yeast S. cerevisiae is highly suitable for this analysis for three reasons. First, yeast tRNAs are modified with dihydrouridine at most of the sites of modification that have been found in characterized tRNAs. Thus, each of the six most common dihydrouridine modification sites are found in multiple yeast cytoplasmic tRNAs (Table I), and two of the five minor dihydrouridine modification sites are found in its sequen...

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Section: Discussionmentioning

confidence: 97%

The Specificities of Four Yeast Dihydrouridine Synthases for Cytoplasmic tRNAs

Xing

Hiley

Hughes

et al. 2004

Journal of Biological Chemistry

100

114

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“…There is no obvious homology with other methyltransferases, based on BLAST search. Moreover, there is little, if any, similarity between Trm13 protein and other tRNA 29-O-methyltransferases identified in yeast (Cavaille et al 1999;Pintard et al 2002a,b;Lapeyre and Purushothaman 2004), as determined by pairwise BLAST, or with the RrmJ/fibrillarin superfamily of 29-Omethyltransferases (Feder et al 2003). Since Trm13 protein is described in the PFAM database as a domain of unknown function (DUF 715) and all of the putative Trm13 protein homologs are annotated as novel or hypothetical proteins, the Trm13 family might represent an additional family of methyltransferase proteins, although the low degree of similarity among methyltransferases within the family precludes a definitive statement in this regard (Schubert et al 2003).…”

Section: Trm13 Protein Is Highly Conserved Within Eukaryotes and Is Nmentioning

confidence: 99%

“…Trm7 methylates the ribose at positions 32 and 34 of three tRNAs, and trm7 mutants have an important role in translational fidelity (Pintard et al 2002b). Trm3 modifies G 18 on 10 of the 34 sequenced tRNAs (Cavaille et al 1999), and, like many other modification proteins that act at remote sites from the anticodon (Hopper and Phizicky 2003), disruption of the gene has little obvious effect on growth or translation (Cavaille et al 1999).…”

Section: Introductionmentioning

confidence: 99%

The 2′-O-methyltransferase responsible for modification of yeast tRNA at position 4

Wilkinson¹,

Crary²,

Jackman³

et al. 2007

RNA

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The methylation of the ribose 29-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 29-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 29-O-methylation that occurs in the middle of a duplex region in tRNA, the modification is conserved in eukaryotes, and the features of the tRNA necessary for substrate recognition are poorly defined. We show here that Saccharomyces cerevisiae ORF YOL125w (TRM13) is necessary and sufficient for 29-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 29-O-methylation activity, using tRNA Gly(GCC) as a substrate, and extracts made from a trm13-D strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-D strain lack the corresponding 29-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 29-O-methylation at position 4 in vitro since yeast Trm13 protein purified after expression in Escherichia coli has the same activity as that produced in yeast. Trm13 protein binds substrates tRNA His and tRNA Gly(GCC) with K D values of 85 6 8 and 100 6 14 nM, respectively, and has a K M for tRNA His of 10 nM, but binds nonsubstrate tRNAs very poorly (K D > 1 mM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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“…The Lhp1 protein has been proposed to function as an RNA chaperone facilitating endonucleolytic trimming of the 39 trailers of pre-tRNAs (Yoo & Wolin, 1997)+ In fact, a mutation in the sup61 gene (sup61-10 ) located at position 28 of the tRNA was previously shown to be lethal in combination with a disruption of the LHP1 gene+ When Lhp1 was depleted from sup61-10 cells, a decrease in partially processed as well as mature tRNA CGA Ser was seen (Yoo & Wolin, 1997)+ The similarity in phenotypes and the fact that a catalytically inactive Trm2p suppressed the phenotype of the sup61-T51C trm2 strain prompted us to investigate the role of Lhp1p in the sup61 mutant strains isolated by us+ The Lhp1 protein has, to our knowledge, no tRNA modification activity+ Moreover, we could not detect any apparent difference in modified nucleosides between a wild-type and a lhp1 strain in HPLC analysis of total tRNA (data not shown)+ The sup61-T51C, sup61-T44G, sup61-T47:2C, and sup61-T20A alleles were combined with a lhp1 disruption and the resulting sup61 lhp1 double mutant strains behaved like the sup61 trm2 strains, that is, viable at 25 8C but inviable or very slow growing at 30 8C (Fig+ 5)+ We also investigated the previously identified sup61-10 allele (Yoo & Wolin, 1997) for genetic interaction with a trm2 null allele and a sup61-10 trm2 double mutant was inviable at 30 8C (data not shown)+ These data indicated similar functions of the Trm2 and Lhp1 proteins with respect to stabilization of tRNA during maturation+ However, a trm2 lhp1 double mutant strain was viable with no obvious growth defect and the processing pattern of tRNA CGA Ser in the trm2 lhp1 strain was identical to that of the single lhp1 mutant (data not shown; Yoo & Wolin, 1997)+ This suggested that the Trm2p and Lhp1p did not have redundant functions, but rather that both proteins were essential in the maturation process of mutant tRNA CGA Ser + It seemed possible that removal of any gene encoding a protein interacting with tRNA CGA Ser in the maturation process would lead to a synergistic phenotype with the various mutant sup61 alleles+ To investigate this hypothesis, the sup61-T51C, sup61-T44G, sup61-T47:2C, and sup61-T20A alleles were combined with null alleles of PUS4, TRM1, and TRM3 encoding pseudo- (Becker et al+, 1997), tRNA (m 2 2 G 26 )dimethyltransferase (Ellis et al+, 1986), and tRNA(Gm 18 ) 29-O-ribose methyltransferase (Cavaille et al+, 1999), respectively+ Precursor tRNA CGA Ser has m 2 2 G 26 and ⌿ 55 whereas Gm 18 is present only in the mature tRNA (Etcheverry et al+, 1979)+ A null allele of the RIT1 gene encoding a tRNA i…”

Section: Mjo Johansson and As Byströmmentioning

confidence: 99%