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. 2007 Mar;13(3):404-13.
doi: 10.1261/rna.399607. Epub 2007 Jan 22.

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

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

Martha L Wilkinson et al. RNA. 2007 Mar.

Abstract

The methylation of the ribose 2'-OH of RNA occurs widely in nature and in all stable RNAs and occurs at five positions in yeast tRNA. 2'-O-methylation of tRNA at position 4 is interesting because it occurs in the acceptor stem (which is normally undermodified), it is the only 2'-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 2'-O-methylation at position 4 of yeast tRNA. Biochemical analysis of the S. cerevisiae proteome shows that Trm13 copurifies with 2'-O-methylation activity, using tRNAGlyGCC as a substrate, and extracts made from a trm13-Delta strain have undetectable levels of this activity. Trm13 is necessary for activity in vivo because tRNAs isolated from a trm13-Delta strain lack the corresponding 2'-O-methylated residue for each of the three known tRNAs with this modification. Trm13 is sufficient for 2'-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 tRNAHis and tRNAGlyGCC with KD values of 85+/-8 and 100+/-14 nM, respectively, and has a KM for tRNAHis of 10 nM, but binds nonsubstrate tRNAs very poorly (KD>1 microM). Trm13 is conserved in eukaryotes, but there is no sequence similarity between Trm13 and other known methyltransferases.

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Figures

FIGURE 1.
FIGURE 1.
Identification of the yeast ORF associated with 2′-O-methylation of tRNAGly(GCC) (A) Schematic of the two-dimensional structure of the tRNAGly(GCC) from S. cerevisiae. The box highlights the 2′-O-methylated base at position four of tRNAHis and tRNAPro. (B) Assay scheme to detect 2′-O-methylation of [α-32P] labeled tRNAGly. After incubation with protein and S-adenosylmethionine, tRNA is treated with RNase T2/RNase A and phosphatase and the products are resolved by thin-layer chromatography. This assay yields Cmp*A if the substrate is modified or Pi* (inorganic phosphate) if the substrate remains unmodified. (C) Assay of genomic collection of purified MORF fusion proteins for 2′-O-methylation activity. Labeled tRNAGly was incubated at 30°C for 18 h in 10 μL reaction mixtures containing S-adenosylmethionine and 2 μL (∼0.7 μg) protein from 59 pools of purified fusion proteins from the MORF collection (numbered 1–53, 55, 56, 58, 59, 64, and 66), as indicated, and analyzed as described in B and Materials and Methods. (a) S. cerevisiae crude extract, 30 μg; (b) buffer control. (D) Assay of subpools from pool 45 for tRNA 2′-O-methylation activity. (First panel) labeled tRNAGly was incubated with 2 μL of pools of purified MORF fusion proteins derived from the strains in rows A–H of plate 45, as indicated. (a) crude extract; (b) buffer. (Second panel) substrate was incubated with MORF fusion proteins from columns 1–12 from plate 45. (a) crude extract; (b) buffer.
FIGURE 2.
FIGURE 2.
Trm13 is required in vitro and in vivo for 2′-O-methylation of tRNA. (A) Extract from a trm13-Δ strain has no detectable 2′-O-methyltransferase activity. A MATα trm13-Δ strain, a control MATα strain (ynr069c-Δ), and a control diploid (TRM13 +/TRM13 +) strain were grown to mid-log phase in YP media containing 2% glucose, and extracts were assayed for 2′-O-methyltransferase activity for 2 h at 30°C with [α-32P] labeled tRNAGly as described in Figure 1, with decreasing amounts of crude extract (by factors of 10, beginning with ∼30 μg protein). (B–D) A trm13-Δ strain lacks 2′-O-methylated residues expected at position 4 of tRNA. tRNAPro (B), tRNAHis (C), and tRNAGly (D) were purified from wild-type and trm13-Δ strains, and their nucleosides were resolved by HPLC as described in Materials and Methods. tRNAs isolated from wild type contain all expected modifications as annotated in the literature (Yoshida 1973; Keith et al. 1983), whereas tRNAs isolated from trm13-Δ strains contain all expected modifications except the 2′-O-methylated residue. Traces shown in the left panel for each tRNA describe a sample modification whose quantification is unaffected by the deletion of TRM13. Traces shown in the right panel correspond to the 2′-O-methylated nucleosides found in each tRNA.
FIGURE 3.
FIGURE 3.
tRNAHis from a trm13-Δ strain lacks 2′-O-methylation at position 4. (A) Clover leaf structure of tRNAHis. The position of the primer used for primer extension analysis is indicated by the arrow. The horizontal arrow indicates the base expected to be 2′-O-methylated. (B) Primer extension to assess 2′-O-methylation of tRNAHis at position 4. Low-molecular-weight RNA derived from wild-type or trm13-Δ strains, as indicated, was analyzed by primer extension using the primer shown in panel A. Lanes A, C, G, T contain the indicated dideoxynucleotide species to generate a sequencing ladder for tRNAHis, with the expected sequence as indicated. The arrow indicates the position of the expected primer extension stop due to the presence of the 2′-O-methylated nucleotide.
FIGURE 4.
FIGURE 4.
Purified His6-Trm13 protein catalyzes formation of 1 mol of 2′-O-methylcytidine at position 4 of tRNAGly in vitro. (A) Schematic of substrate tRNAGly bearing a single labeled 32P between C4 and A5 (C4*-tRNAGly). (B) His6-Trm13 protein purified from E. coli catalyzes 2′-O-methylation of residue 4 of tRNAGly. Reaction mixtures containing a labeled C4*-tRNAGly substrate and decreasing concentrations of purified His6-Trm13 protein (by factors of 10, beginning with 0.68 mg/mL) were incubated for 1.5 h at 30°C, and then RNA was analyzed as described in Materials and Methods. (C) His6-Trm13 protein catalyzes formation of 1 mol of 2′-O-methylcytidine per mole of tRNAGly. His6-Trm13 protein (3.2 μM) or buffer was incubated with 10 μM (2 nmol) unlabeled transcribed tRNAGly for 2 h, and RNA was treated with P1 nuclease and phosphatase to form nucleosides, which were resolved by HPLC. The extra product formed with Trm13 protein matches the accepted retention time and UV spectrum for 2′-O-methylcytidine (Gehrke and Kuo 1989). The expanded area shows a high-resolution view of the region of the HPLC trace corresponding to 2′-O-methylcytidine.
FIGURE 5.
FIGURE 5.
Trm13 protein binds tRNA substrates with higher affinity than nonsubstrate tRNAs. (A,B) Analysis of binding by EMSA. tRNAHis (0.3 nM) and tRNAPhe (0.3 nM) were incubated with varying amounts of His6-Trm13 protein in the presence of 10 μg/mL poly(A) RNA and electrophoresed on a 5% acrylamide gel as described in Materials and Methods. His6-Trm13 protein varied from 2 μM to 0.9 nM (by factors of 3). The first lane in each panel contains no added Trm13 protein. (C) Determination of binding constants. The fraction of total RNA bound in each EMSA assay as shown in panel A was quantified by a PhosphorImager and plotted as a function of Trm13 protein concentration (-◊-, tRNAGly; -•-, tRNAHis; -□-, tRNALeu; and -▴-, tRNAPhe). For tRNAHis and tRNAGly, the resulting data were well fit by a single binding isotherm, and apparent binding constants were determined to be 85 ± 8 nM for tRNAHis and 100 ± 14 nM for tRNAGly. Since little or no apparent binding is observed for tRNALeu and tRNAPhe at even the highest protein concentrations tested, we can estimate lower limits to the KD for these tRNA species of >1 μM and >3 μM, respectively.
FIGURE 6.
FIGURE 6.
Kinetic parameters for His6-Trm13 2′-O-methyltransferase activity with tRNAHis. His6-Trm13 protein (0.5 nM) was incubated with C3*-tRNAHis at different concentrations (5 nM, 10 nM, 20 nM, 50 nM, and 100 nM) for 2.5–20 min at 30°C and analyzed for 2′-O-methylation by TLC as described in Materials and Methods. (A) Representative time course of 2′-O-methyltransfease activity of His6-Trm13 protein with tRNAHis. 2′-O-methyltransferase activity was assayed with 0.5 nM Trm13 and 5 nM tRNAHis at (lane a) 2.5 min, (lane b) 5 min, (lane c) 10 min, (lane d) 20 min; (lane e) the results of reaction with 100 nM tRNAHis and 920 nM His6-Trm13 protein for 20 min, showing that all the substrate can react; (lane f) 100 nM tRNAHis with no Trm13 protein for 20 min. (B) Determination of steady-state kinetic parameters for Trm13 2′-O-methyltransferase activity with tRNAHis. Trm13-catalyzed 2′-O-methylation of tRNAHis occurs with a steady-state kcat = 0.24 min−1, KM = 10 nM, and kcat/KM = 4.0 × 105 M−1sec−1, as determined by fit of the data to the Michealis–Menten steady-state rate equation (Materials and Methods).
FIGURE 7.
FIGURE 7.
Alignment of Trm13 homologs. Homologs of Trm13 were identified by BLAST search (Altschul et al. 1997) and sequences from several representative species, as indicated next to each sequence line, were aligned using Multalin (Corpet 1988) with the following parameters: gap weight = 6 and gap length weight = 2. Dark shading indicates >80% consensus; light shading indicates >50% consensus at each position. For clarity, several regions with very low homology were omitted from this alignment; these breaks in the sequence are indicated with double hash marks. The numbering shown above each block of sequence represents the numbering of the relevant S. cerevisiae protein residue.

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