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. 2015 Dec 15;43(22):10952-62.
doi: 10.1093/nar/gkv980. Epub 2015 Sep 30.

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Martin Müller  1 , Mark Hartmann  2 , Isabelle Schuster  3 , Sebastian Bender  2 , Kathrin L Thüring  4 , Mark Helm  4 , Jon R Katze  5 , Wolfgang Nellen  3 , Frank Lyko  2 , Ann E Ehrenhofer-Murray  6
Affiliations

Dynamic modulation of Dnmt2-dependent tRNA methylation by the micronutrient queuine

Martin Müller et al. Nucleic Acids Res. .

Abstract

Dnmt2 enzymes are cytosine-5 methyltransferases that methylate C38 of several tRNAs. We report here that the activities of two Dnmt2 homologs, Pmt1 from Schizosaccharomyces pombe and DnmA from Dictyostelium discoideum, are strongly stimulated by prior queuosine (Q) modification of the substrate tRNA. In vivo tRNA methylation levels were stimulated by growth of cells in queuine-containing medium; in vitro Pmt1 activity was enhanced on Q-containing RNA; and queuine-stimulated in vivo methylation was abrogated by the absence of the enzyme that inserts queuine into tRNA, eukaryotic tRNA-guanine transglycosylase. Global analysis of tRNA methylation in S. pombe showed a striking selectivity of Pmt1 for tRNA(Asp) methylation, which distinguishes Pmt1 from other Dnmt2 homologs. The present analysis also revealed a novel Pmt1- and Q-independent tRNA methylation site in S. pombe, C34 of tRNA(Pro). Notably, queuine is a micronutrient that is scavenged by higher eukaryotes from the diet and gut microflora. This work therefore reveals an unanticipated route by which the environment can modulate tRNA modification in an organism.

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Figures

Figure 1.
Figure 1.
In vivo methylation of tRNAAsp by the Dnmt2 homologs Pmt1 (Schizosaccharomyces pombe) and DnmA (Dictyostelium discoideum) was stimulated by growth of cells in the presence of queuine. (A) In vitro methylation of total RNA from S. pombe (left) and D. discoideum strains (right) that were either wild-type (wt), lacked Dnmt2 (pmt1Δ, dnmA-) or overexpressed DnmA (dnmA-GFP) and were grown in the indicated medium (S.pombe: full—YES; D. discoideum: full—HL5, minimal—FM) with or without queuine (0.03 μM in S. pombe; 0.1 μM in D. discoideum). Methylation was performed by recombinant Pmt1 (left) or DnmA (right). Top, ethidium bromide-stained gel (EtBr; size of ribosomal and tRNAs indicated), bottom, autoradiogram of methylated RNA (3H). (B) In vivo RNA bisulfite sequencing of tRNAAsp from total RNA of wild-type S. pombe grown with or without queuine (Q) as described in (A). The cytosine residues present in tRNAAsp are indicated in the top row. Each subsequent row represents an independent clone that was sequenced. Black boxes indicate methylated cytosines, white boxes indicate unmethylated cytosines, the arrow indicates the methylation position for Dnmt2 enzymes. Data for wt are taken from (14). (C) In vivo RNA bisulfite sequencing of tRNAAsp from total RNA of D. discoideum, representation as in (B).
Figure 2.
Figure 2.
Queuine incorporation into tRNA in vivo stimulated Dnmt2 enzymes in vivo and in vitro. (A) Quantitation of queuine levels in total RNA from Schizosaccharomyces pombe (wt and pmt1Δ) grown with or without queuine, as determined by LC-MS/MS. Extracted peak areas for queuine were normalized to the UV peak area of guanosine. (B) Pmt1 activity in vitro was stimulated on tRNA containing queuine (Q34) as compared to Q-free tRNA (G34). A time course of methylation by Pmt1 (1 μM) is shown. The left panel shows the time course of incorporation of 3H-labeled methyl groups into total RNA isolated from pmt1Δ cells grown with (Q34) or without queuine (G34). Methylation was quantitated by densitometric analysis of autoradiograms. Right panel, in vitro methylation of tRNAAsp isolated ex vivo from pmt1Δ with or without queuine. Representation and data analysis as in the left panel. Time courses of methylation were reproduced with an independent batch with comparable results. (C) In vivo stimulation of Pmt1 required incorporation of queuine into tRNA by the eukaryotic tRNA-guanine transglycosylase (eTGT). High-throughput bisulfite sequencing of tRNAAsp from strains that were wild-type or lacked Qtrt1, Qtrtd1 or both grown in full medium (YES) with or without queuine as indicated was performed. Results from 1024 independent sequences are shown. Yellow, unmethylated cytosine; blue, methylated cytosine; red, mismatch. Arrows indicate the C38 position. Methylation levels are given in %. (D) DnmA activity in vivo on RNA required the presence of eTGT in Dictyostelium discoideum. Total RNA from the wild-type and two independent qtrt1- mutant strains grown with or without queuine was methylated in vitro by recombinant DnmA. Presentation as in Figure 1A.
Figure 3.
Figure 3.
In vivo methylation of C38-containing tRNAs other than tRNAAsp was not stimulated by queuine. (A) Cytosine methylation levels of tRNAAsp, tRNAHis, tRNAGlu (TTC) and tRNAVal (TAC) in Schizosaccharomyces pombe as determined by whole tRNA bisulfite sequencing are shown. Wild-type cells were grown with or without queuine as indicated. Yellow, unmethylated cytosine; blue, methylated cytosine; red, mismatch. The arrow indicates the C38 position, values for methylation levels are indicated above. For tRNAHis, tRNAGlu and tRNAVal, this corresponds to background values that were also obtained in the absence of queuine or Pmt1 (see Supplementary Table S1). (B) Methylation of mitochondrial tRNAHis, tRNAGlu and tRNAGly was not stimulated by growth in queuine in Dictyostelium discoideum. Analysis as in Figure 1C.
Figure 4.
Figure 4.
Genome-wide high-throughput tRNA bisulfite sequencing in Schizosaccharomyces pombe reveals novel candidate tRNA methylation sites and strong selectivity of Pmt1 for tRNAAsp. (A) Left, cytosine methylation levels are shown for individual tRNA species in wt cells grown with queuine. The top row indicates the position of the cytosine using standard tRNA numbering. Blue, strong methylation; yellow, no methylation. C38 of tRNAAsp and C11 of tRNASer (GCT) are boxed in red/ black for clarity. Novel candidate methylation sites (C61–C63 of tRNAGly and C34 of tRNAPro) are highlighted with an arrow. Middle, difference in tRNA methylation between wt and pmt1Δ cells grown in the presence of queuine. Green colors indicate a strong loss of methylation in pmt1Δ compared to wt. Red colors indicate an increase in methylation in pmt1Δ compared to wt. Right, difference in tRNA methylation between wt cells cultivated with and without queuine (ex Q, cells were grown in YES). Data is represented as in the middle panel. (B) Methylation levels of the indicated tRNAs in wt and pmt1Δ cells grown with or without queuine. tRNASer (GCT) methylation was mildly increased in the absence of Pmt1 and queuine.
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