Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9 - PubMed (original) (raw)

Identification of the yeast gene encoding the tRNA m1G methyltransferase responsible for modification at position 9

Jane E Jackman et al. RNA. 2003 May.

Abstract

Methylation of tRNA at the N-1 position of guanosine to form m(1)G occurs widely in nature. It occurs at position 37 in tRNAs from all three kingdoms, and the methyltransferase that catalyzes this reaction is known from previous work of others to be critically important for cell growth in Escherichia coli and the yeast Saccharomyces cerevisiae. m(1)G is also widely found at position 9 in eukaryotic tRNAs, but the corresponding methyltransferase was unknown. We have used a biochemical genomics approach with a collection of purified yeast GST-ORF fusion proteins to show that m(1)G(9) formation of yeast tRNA(Gly) is associated with ORF YOL093w, named TRM10. Extracts lacking Trm10p have undetectable levels of m(1)G(9) methyltransferase activity but retain normal m(1)G(37) methyltransferase activity. Yeast Trm10p purified from E. coli quantitatively modifies the G(9) position of tRNA(Gly) in an S-adenosylmethionine-dependent fashion. Trm10p is responsible in vivo for most if not all m(1)G(9) modification of tRNAs, based on two results: tRNA(Gly) purified from a trm10-Delta/trm10-Delta strain is lacking detectable m(1)G; and a primer extension block occurring at m(1)G(9) is removed in trm10-Delta/trm10-Delta-derived tRNAs for all 9 m(1)G(9)-containing species that were testable by this method. There is no obvious growth defect of trm10-Delta/trm10-Delta strains. Trm10p bears no detectable resemblance to the yeast m(1)G(37) methyltransferase, Trm5p, or its orthologs. Trm10p homologs are found widely in eukaryotes and many archaea, with multiple homologs in several metazoans, including at least three in humans.

PubMed Disclaimer

Figures

FIGURE 1.

FIGURE 1.

(A) N1-Methylguanosine. (B) Assay scheme to detect m1G formation in tRNAGly uniquely labeled at position 9 (G9*Gly). This assay results in the production of either Gp* if the substrate remains unmodified, or m1Gp* if it is modified.

FIGURE 2.

FIGURE 2.

Identification of a yeast ORF associated with m1G9 methyltransferase activity. Reaction mixtures containing G9*Gly tRNA and protein as indicated were incubated in methyltransferase buffer at 30°C for 4 h, and then RNA was digested with nucleases to produce 3′-phosphorylated nucleotides, which were resolved by thin layer chromatography. (A) Assay of a genomic collection of pools of purified yeast GST-ORF fusion proteins for G9 methyltransferase activity. Substrate G9*Gly tRNA was incubated with 64 pools of purified GST-ORF fusion proteins, each derived from 96 yeast strains of a library of strains expressing individual GST-ORF fusion proteins, as indicated. (Lane a) Saccharomyces cerevisiae crude extract; (lane b) no extract. (B) Assay of subpools from pool 47 for tRNA G9 methyltransferase activity. (First panel) Substrate G9*Gly tRNA was incubated with pools of GST-ORFs derived from the strains in rows A_–_H from plate 47, as indicated. (Lane P47) plate 47; (lane a) crude extract; and (lane b) no extract. (Second panel) Substrate was incubated with GST-ORFs from columns 1–12 of plate 47. (Lanes P47,a,b,) Same as previous panel.

FIGURE 3.

FIGURE 3.

Assay of a _trm10-_Δ/_trm10-_Δ strain for methyltransferase activity at positions 9 and 37. Extracts were prepared from isogenic wild-type (TRM10+/TRM10+) and _trm10-_Δ/_trm10-_Δ strains, and assayed for methyltransferase activites. (A) m1G9 methyltransferase activity. Assays contained G9*Gly tRNA and decreasing concentrations of crude extracts (5 mg/mL to 1.6 μg/mL by factors of 5) made from wild-type and _trm10-_Δ/_trm10-_Δ yeast strains and were carried out at 30°C for 1 h. (B) m1G37 methyltransferase activity. Assays contained G37*Leu tRNA with decreasing concentrations of crude extracts from wild-type and _trm10-_Δ/_trm10-_Δ yeast strains (2.5 mg/mL to 2.5 μg/mL by factors of 10) and were carried out at 30°C for 5 h.

FIGURE 4.

FIGURE 4.

Overexpression and purification of Trm10p from Escherichia coli. (A) SDS-PAGE gel of Trm10p overproduction and purification. (Lane 1) Crude extract from control strain with vector only (40 μg); (lane 2) crude extract from strain with plasmid pJEJ12-3 expressing His6-Trm10p (40 μg); (lane 3) broad range MW markers; (lane 4) crude extract used for purification, from strain containing pJEJ12-3 (40 μg); (lanes 5,6) purified Trm10p (4 and 8 μg, respectively). (B) Assay of E. coli extracts for m1G9 methyltransferase activity. Assays containing G_9_*Gly tRNA and decreasing concentrations of either the control extract (panel A, lane 1) or Trm10p-expressing extract (panel A, lane 2) were performed at 30°C for 1 h.

FIGURE 5.

FIGURE 5.

Identification of m1G as the product of Trm10p activity in vitro and in vivo. (A) Trm10 protein modifies tRNAGly to produce material that matches m1G. (Upper trace) m1G chemical standard. (Middle trace) In vitro transcribed tRNAGly treated with buffer and digested to nucleosides. (Lower trace) In vitro transcribed tRNAGly treated with Trm10p and digested to nucleosides. All samples were analyzed by HPLC as described in Materials and Methods. (B) Comparison of nucleosides of tRNAGly from wild-type and _trm10-_Δ/_trm10-_Δ strains. tRNAGly was purified from wild-type and trm10 mutant strains, and its nucleosides were prepared and resolved on HPLC as described above.

FIGURE 6.

FIGURE 6.

Deletion of TRM10 results in loss of m1G9, but not m1G37 in vivo. (A) Establishment of primer extension assay to assess m1G modification. (Left panel) Cloverleaf structure of tRNAGly. Nucleotides in bold indicate the position of the primer used for primer extension analysis of the 5′-end of the tRNA. (Right panel) Primer was used with in vitro transcribed tRNAGly and AMV Reverse Transcriptase to generate a sequencing ladder (lanes G, A, T, and C), and alone (−) or with RNA derived from wild type (wt) or _trm10-_Δ/_trm10-_Δ (Δ) strains to analyze for the presence of m1G9 in tRNAGly from each population of RNA. (B) Primer extension to assess modification status of tRNAVal, tRNATrp, tRNAICGArg, and tRNAUUULys at position G9. Primer extensions were performed with either (−) primer alone, (wt) wild-type RNA, or (Δ) _trm10-_Δ/_trm10-_Δ RNA. (C) Primer extension to assess modification status of tRNAUAGLeu and tRNAPhe at position G37. Same lanes as B.

FIGURE 7.

FIGURE 7.

Alignment of Trm10p homologs. The various Trm10 homologs were identified using iterative BLAST searches, and aligned using CLUSTALX. A representative alignment is shaded to a 50% consensus using MacBoxShade, and indicates residues that are similar (gray background) or identical across all species (black background). The numbers adjacent to the name of each species are the accession numbers for the proteins used. The numbers in parentheses before and after the alignment indicate the numbers of amino acids from individual sequences not included in the alignment for clarity of presentation.

FIGURE 8.

FIGURE 8.

Phylogeny of the Trm10p homologs. A phylogeny of the various Trm10p homologs from eukaryotes and archaea was constructed using the neighbor-joining method, rooted on the archaeal homologs. Bootstrap analysis was carried out using the PAUP* version 4.0b10, and bootstrap support (percentage of 1000 trials where a certain grouping was supported) is indicated at the respective nodes. The tree is collapsed to a 50% bootstrap support. The source species of each protein is listed beside the appropriate line, together with the corresponding accession number.

References

    1. Alexandrov, A.V., Martzen, M.R., and Phizicky, E.M. 2002. Two proteins that form a complex are required for 7-methylguanosine modification of yeast tRNA. RNA 8: 1253–1266. - PMC - PubMed
    1. Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 25: 3389–3402. - PMC - PubMed
    1. Ansmant, I., Motorin, Y., Massenet, S., Grosjean, H., and Branlant, C. 2001. Identification and characterization of the tRNA:Psi 31-synthase (Pus6p) of Saccharomyces cerevisiae. J. Biol. Chem. 276: 34934–34940. - PubMed
    1. Basavappa, R. and Sigler, P. 1991. The 3 Å crystal structure of yeast indicator tRNA: Functional implications in initiator/elongator discrimination. EMBO J. 10: 3105–3111. - PMC - PubMed
    1. Becker, H.F., Motorin, Y., Planta, R.J., and Grosjean, H. 1997. The yeast gene YNL292w encodes a pseudouridine synthase (Pus4) catalyzing the formation of psi55 in both mitochondrial and cytoplasmic tRNAs. Nucleic Acids Res. 25: 4493–4499. - PMC - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources