Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press tRNA m7G methyltransferase Trm8p/Trm82p: Evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p ANDREI ALEXANDROV, ELIZABETH J. GRAYHACK, and ERIC M. PHIZICKY Department of Biochemistry and Biophysics, University of Rochester School of Medicine, Rochester, New York 14642, USA ABSTRACT We show that Saccharomyces cerevisiae strains lacking Trm8p/Trm82p tRNA m7G methyltransferase are temperature-sensitive in synthetic media containing glycerol. Bacterial TRM8 orthologs complement the growth defect of trm8-⌬, trm82-⌬, and trm8-⌬ trm82-⌬ double mutants, suggesting that bacteria employ a single subunit for Trm8p/Trm82p function. The growth phenotype of trm8 mutants correlates with lack of tRNA m7G methyltransferase activity in vitro and in vivo, based on analysis of 10 mutant alleles of trm8 and bacterial orthologs, and suggests that m7G modification is the cellular function important for growth. Initial examination of the roles of the yeast subunits shows that Trm8p has most of the functions required to effect m7G modification, and that a major role of Trm82p is to maintain cellular levels of Trm8p. Trm8p efficiently cross-links to pre- tRNAPhe in vitro in the presence or absence of Trm82p, in addition to its known residual tRNA m7G modification activity and its SAM-binding domain. Surprisingly, the levels of Trm8p, but not its mRNA, are severely reduced in a trm82-⌬ strain. Although Trm8p can be produced in the absence of Trm82p by deliberate overproduction, the resulting protein is inactive, suggesting that a second role of Trm82p is to stabilize Trm8p in an active conformation. Keywords: tRNA modification; S. cerevisiae; S-adenosylmethionine; methyltransferases; Trm8; Trm82; YggH; TM0925; 7-methylguanosine; m7G INTRODUCTION Gcd14p (Anderson et al. 1998), I34 adenosine deaminase Tad2p/Tad3p (Gerber and Keller 1999), and tRNAHis G A continuing puzzle in RNA biology is the precise role of −1 guanylyltransferase Thg1p (Gu et al. 2003). Strains lacking tRNA modifications. Although more than 79 different each of six other modification enzymes affecting residues in modifications have been identified in tRNA (Limbach et al. the anticodon region have distinct growth or translation 1994; Bjork 1995), and 25 of these modifications are found phenotypes, including 2Ј-O-Me methyltransferase in yeast (Sprinzl et al. 1998), a cellular role for many of 32,34 Trm7p (Pintard et al. 2002), m1G /m1I methyltransfer- them has not yet been defined. The recent identification of 37 37 ase Trm5p (Bjork et al. 2001), ⌿ pseudouridylase a substantial number of genes responsible for tRNA modi- 38,39 Pus3p (Lecointe et al. 1998), mcm5U/mcm5s2U carboxyl fication (Hopper and Phizicky 2003) has allowed the op- 34 methyltransferase Trm9p (Kalhor and Clarke 2003), portunity to study their roles by examining mutant pheno- m5C methyltransferase Trm4p (Wu et al. 1998), types. 34,40,48,49 and i6A isopentenyl transferase Mod5p (Laten et al. 1978; Surprisingly, deletion of the majority of enzymes respon- 37 Janner et al. 1980; Dihanich et al. 1987). sible for tRNA modification revealed few obvious defects. The other 16 known modification enzymes in yeast each Only three tRNA modifying enzymes in yeast are known to modify residues remote from the anticodon region, and be essential, including m1A methyltransferase Gcd10p/ 58 mutants lacking these enzymes have only subtle pheno- types. Each of these mutants has little obvious growth or Reprint requests to: Eric Phizicky, Department of Biochemistry and translation defect, and the observation of distinct pheno- Biophysics, University of Rochester School of Medicine, 601 Elmwood types has required more sensitive approaches such as syn- Ave., Box 712, Rochester, NY 14642, USA; e-mail eric_phizicky@ thetic interaction screens (Grosshans et al. 2001; Johansson urmc.rochester.edu. Article published online ahead of print. Article and publication date are and Bystrom 2002; Urbonavicius et al. 2002), or, as shown at http://www.rnajournal.org/cgi/doi/10.1261/rna.2030705. in Escherichia coli, growth competition experiments (Gutg- RNA (2005), 11:821–830. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 RNA Society. 821 Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press Alexandrov et al. sell et al. 2000). The lack of obvious phenotype of strains Aquifex aeolicus aq065 protein purified from E. coli (Oka- lacking these modification enzymes contrasts sharply with moto et al. 2004). the strong evolutionary conservation of a number of these As a first step toward determining the physiological roles enzymes and their modifications. Even more unexpectedly, of TRM8/TRM82 genes and the role of m7G modification of some of the subtle phenotypes associated with defects in tRNA, we have found and characterized a phenotype asso- 5 tRNA modification, including those of yeast m U54 meth- ciated with loss of either or both of the TRM8 and TRM82 yltransferase Trm2p (Johansson and Bystrom 2002) and E. genes. We show that this growth phenotype is strongly cor- ⌿ 7 coli 55 pseudouridylase TruB (Gutgsell et al. 2000), were related with m G methyltransferase activity in vitro and in complemented with catalytically inactive mutants, suggest- vivo. We also provide in vivo evidence that the bacterial ing that lack of the modification of tRNA is not the cause of Trm8 ortholog does not require a second subunit to func- the defect, and that tRNA-modifying enzymes may have tion in yeast, raising the issue of the function of each of the other distinct cellular roles. two yeast subunits. We focus here on m7G (7-methylguanosine) modifica- We provide evidence that a crucial function of yeast tion of tRNA. This modification is highly conserved, based Trm82p is to maintain the levels of active Trm8p in vivo, on its presence in >40% of all sequenced bacterial and since deletion of Trm82p results in a severe reduction in the eukaryotic tRNA species. In all but three cases, m7Gis level of Trm8p protein, and Trm8p prepared from yeast in found at position 46 in the extra loop, a site known from the absence of Trm82p is not detectably active. In contrast, the crystal structures of tRNAPhe (Kim et al. 1974; Robertus Trm8p appears to possess the requisite functions of the Met et al. 1974) and tRNAi (Basavappa and Sigler 1991) to catalytic subunit: it has a SAM-binding domain, has re- form tertiary interactions with bases at positions 13 and 22. sidual catalytic activity when purified from E. coli, and Additionally, the m7G and m1A modification are the only cross-links to pre-tRNAPhe in vitro. two yeast modifications that confer a positive charge to the base. We previously used a biochemical genomics approach to RESULTS identify two proteins, Trm8p and Trm82p, that copurify with tRNA m7G methyltransferase activity. We showed that TRM8 and TRM82 are both required for growth at 7 these two proteins are both necessary for m G46 modifica- 38°C on glycerol-containing synthetic media tion of tRNA in vivo and in vitro, form a complex in vivo, We found a distinct growth defect that results from the loss and are sufficient for activity in vitro (Alexandrov et al. of TRM8 or TRM82 genes. As demonstrated in Figure 1 by 2002). Neither Trm8p nor Trm82p is significantly related to 7 serial 10-fold dilutions of yeast cells, homozygous diploid yeast Abd1p, which catalyzes m G formation during cap- strains lacking either Trm8p (relevant genotype: trm8-⌬/ ping of mRNAs (Mao et al. 1995), other than within the trm8-⌬) or Trm82p (relevant genotype: trm82-⌬/trm82-⌬) S-adenosylmethionine (SAM)-binding domain of Trm8p. grow extremely poorly relative to wild-type strains on syn- Trm8p is highly conserved in eukaryotes and bacteria, ex- thetic minimal media (Sherman 1991) containing 2% glyc- tending from humans to Mycoplasma genitalium, with less erol at 38°C. No obvious growth differences were observed that 500 ORFs (Bahr et al. 1999), and Trm82p orthologs are between mutants and the wild-type strain on this media at present in the majority of sequenced eukaryotes. Neverthe- 30°Corat36°C, or on two other media at either 30°Cor less, deletion of TRM8 or TRM82 results in no obvious growth defect under standard laboratory conditions (Alex- androv et al. 2002), raising the question of the role of these proteins in these organisms. Surprisingly, whereas Trm8p/Trm82p complex appears to be conserved in eukaryotes, it may be a single Trm8p subunit in bacteria, since bacteria appear to lack Trm82p (Michaud et al. 2000). We showed previously that the two- protein mechanism of tRNA m7G formation is conserved in higher eukaryotes, since expression of both human ortholo- gous proteins (METTL1 and WDR4) in yeast was required to restore m7G methyltransferase activity in extracts (Alex- FIGURE 1. Growth defect of trm8-⌬/trm8-⌬ and trm82-⌬/trm82-⌬ androv et al. 2002). However, it appears that a single bac- strains on glycerol-containing minimal media. Homozygous diploid terial protein may be sufficient for activity, consistent with trm8-⌬/trm8-⌬, trm82-⌬/trm82-⌬, or wild-type strains, containing a the implication of a single purified 25-kDa Salmonella ty- CEN URA3 plasmid bearing TRM8 or TRM82 under control of its own phimurium polypeptide in tRNA m7G methylation (Col- promoter as indicated, were grown overnight, and 10-fold serial dilu- 7 tions were plated on synthetic media lacking uracil. (A) Cells plated on onna et al. 1983), and the demonstration of tRNA m G- glucose-containing media, incubated for2dat30°C. (B) Cells plated modifying activity for E. coli YggH (De Bie et al. 2003), and on glycerol-containing media, incubated for7dat38°C. 822 RNA, Vol. 11, No. 5 Downloaded from rnajournal.cshlp.org on September 28, 2021 - Published by Cold Spring Harbor Laboratory Press tRNA m7G methyltransferase activity and phenotype 38°C: synthetic minimal media containing 2% dextrose, and pendence of the growth defects of trm8-⌬ and trm82-⌬ yeast extract-peptone media containing 2% glycerol (data strains, and the absence of an additional effect in the trm8-⌬ not shown).