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. 2005 May;11(5):821-30.
doi: 10.1261/rna.2030705. Epub 2005 Apr 5.

tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

Andrei Alexandrov  1 , Elizabeth J Grayhack, Eric M Phizicky
Affiliations

tRNA m7G methyltransferase Trm8p/Trm82p: evidence linking activity to a growth phenotype and implicating Trm82p in maintaining levels of active Trm8p

Andrei Alexandrov et al. RNA. 2005 May.

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-Delta, trm82-Delta, and trm8-Delta trm82-Delta 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-Delta 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.

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Figures

FIGURE 1.
FIGURE 1.
Growth defect of trm8-Δ/trm8-Δ and trm82-Δ/trm82-Δ strains on glycerol-containing minimal media. Homozygous diploid trm8-Δ/trm8-Δ, trm82-Δ/trm82-Δ, or wild-type strains, containing a CEN URA3 plasmid bearing TRM8 or TRM82 under control of its own promoter as indicated, were grown overnight, and 10-fold serial dilutions were plated on synthetic media lacking uracil. (A) Cells plated on glucose-containing media, incubated for 2 d at 30°C. (B) Cells plated on glycerol-containing media, incubated for 7 d at 38°C.
FIGURE 2.
FIGURE 2.
Complementation of the growth defect by variants of TRM8 and of two of its bacterial orthologs. Homozygous diploid strains with genetic background indicated at the top of each titration panel, and containing plasmids expressing GST-fusion proteins under PCUP1 control as indicated at the left, were grown overnight and plated after serial 10-fold dilutions, on synthetic media lacking leucine and uracil, and containing carbon sources as indicated. (A) Cells plated on media containing glycerol, and incubated at 38°C for 7 d. (B) Cells plated on media containing glucose, and incubated at 30°C for 3 d. Rows 1a–1e, plasmids expressing variants of yeast TRM8, as described in the text, as GST-fusion proteins; 2, plasmid expressing TRM82; 3a–3c, plasmids expressing E. coli YggH variants; 4a–4c, plasmids expressing T. maritima TM0925 variants; 5, vector control; 6, wild-type strain control, with vector control.
FIGURE 3.
FIGURE 3.
m7G-methyltransferase activity is altered in cells expressing Trm8p, YggH, and TM0925 mutant variants. (A) Analysis of m7G methyltransferase activity of variants. Activity was assayed with [α-32P]GTP-labeled yeast pre-tRNAPhe substrate and fivefold dilutions of crude extracts beginning with 6 μg of protein, and modified nucleotides were analyzed as described in Materials and Methods. Open arrows, m7G methyltransferase activity; black arrows, m22G methyltransferase activity, as internal control. (B) SDS-PAGE analysis of expressed GST fusion proteins. Expressed GST-fusion proteins were purified by glutathione sepharose chromatography from extracts of yeast strains shown in Figures 2 ▶ and 3A ▶, analyzed by 4%–15% SDS-PAGE, and stained with Coomassie. Arrows indicate protein bands of the expected size. (C) Alignment of Motif I and Post I for putative SAM-binding domains of Trm8p-orthologous proteins. Sites of point mutations used in this study (G103, G105, and G124) are indicated with triangles.
FIGURE 4.
FIGURE 4.
Trm8p protein, but not mRNA, is severely reduced in a trm82-Δ strain. (A) Comparison of Trm8p expression in a trm82-Δ and a TRM82+ strain. Otherwise isogenic haploid strains containing wild-type TRM8 or chromosomally epitope-tagged TRM8 (tagged with His6-HA-3Cprotease site-ZZProtein A, abbreviated ZZ), and TRM82+ or trm82-Δ as indicated, were grown, harvested, and lysed with SDS and glass beads as described in Materials and Methods. Lysates were serially diluted fourfold, starting with 2.7 OD600 units of cells, resolved by SDS-PAGE, and analyzed for Trm8-ZZ expression by Western blot analysis, as described in Materials and Methods. Lanes a–c, TRM8-ZZ TRM82+ strain; d–f, TRM8-ZZ trm82-Δ strain; g–i, TRM8-ZZ trm82-Δ strain transformed with plasmid containing CEN LEU2 PTRM82TRM82; j–l, TRM8 TRM82+ strain, with no epitope tag. (B) SDS-PAGE analysis of lysates described in (A) and stained with Coomassie. (C) Comparison of TRM8 mRNA in a trm82-Δ and a TRM82+ strain. RNA from otherwise isogenic wild-type (lanes a–c), trm82-Δ (d–f), and trm8-Δ (g–i) haploid strains was resolved on a 1% agarose gel, transferred to membrane, and hybridized with 32P-labeled TRM8 or ACT1-specific probes, as described in Materials and Methods. Successive lanes contain 15, 7.5, and 3.8 μg RNA.
FIGURE 5.
FIGURE 5.
Trm8p overexpressed in a trm82-Δ strain has much reduced activity. (A) GST-Trm8p or GST alone (mock) were overex-pressed under PCUP1 control in a trm82-Δ or a TRM82+ strain as indicated, and expressed proteins were purified from extracts by chromatography on glutathione sepharose resin, followed by release of the Trm8p moiety by cleavage with GST-3C protease, and then analyzed by 4%–15% SDS PAGE and Coomassie staining. (B) Analysis of m7G methyltransferase activity of purified Trm8p. Trm8p purified as described in A from a trm82-Δ (lanes d–f) or a TRM82+ strain (j–l), or mock-purified controls (lanes g–i, m–o), was analyzed for methyltransferase activity, using serial fivefold dilutions of preparations. Controls: a, yeast m22G tRNA methyltransferase, b, yeast Trm8p/Trm82p m7G methyltransferase, c, buffer only.
FIGURE 6.
FIGURE 6.
Trm8p cross-links to pre-tRNAPhe in the presence and absence of Trm82p. Preparations of yeast Trm82p, Trm8p, Trm8p/ Trm82p, or similarly purified control protein 3C protease were subjected to UV cross-linking with pre-tRNAPhe as described in Materials and Methods, followed by RNase treatment and resolution by 10% SDS-PAGE. (A) Analysis of proteins after SDS-PAGE and Coomassie staining. (B) Analysis of radioactivity incorporated in gel shown in A.
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