Pmt1, a Dnmt2 homolog in Schizosaccharomyces pombe, mediates tRNA methylation in response to nutrient signaling

Abstract

The fission yeast Schizosaccharomyces pombe carries a cytosine 5-methyltransferase homolog of the Dnmt2 family (termed pombe methyltransferase 1, Pmt1), but contains no detectable DNA methylation. Here, we found that Pmt1, like other Dnmt2 homologs, has in vitro methylation activity on cytosine 38 of tRNA(Asp) and, to a lesser extent, of tRNA(Glu), despite the fact that it contains a non-consensus residue in catalytic motif IV as compared with its homologs. In vivo tRNA methylation also required Pmt1. Unexpectedly, however, its in vivo activity showed a strong dependence on the nutritional status of the cell because Pmt1-dependent tRNA methylation was induced in cells grown in the presence of peptone or with glutamate as a nitrogen source. Furthermore, this induction required the serine/threonine kinase Sck2, but not the kinases Sck1, Pka1 or Tor1 and was independent of glucose signaling. Taken together, this work reveals a novel connection between nutrient signaling and tRNA methylation that thus may link tRNA methylation to processes downstream of nutrient signaling like ribosome biogenesis and translation initiation.

Figure 1.

Pmt1 has in vitro tRNA methylation activity. (A) in vitro methylation activity of Pmt1 and its homologs DnmA (D. discoideum) and human Dnmt2 on in vitro-transcribed tRNA^Asp from D. discoideum (D.d.). Methylation assays were performed using ³H-labeled AdoMet with 3 and 12 µM Pmt1 or 3 µM each for DnmA and hDnmt2 on 500 ng of in vitro-transcribed tRNA^Asp or tRNA^AspC38A from D. discoideum. Samples were separated by urea-PAGE, and methylation was detected by autoradiography. (B) Pmt1 activity on in vitro-transcribed tRNA^Asp from S. pombe. Pmt1 activity was abrogated by mutation of cysteine 81 within catalytic motif IV (C81A). Assays were performed as in A. (C) Coomassie-stained SDS gel of recombinant Dnmt2 homologs used in A and B). (D) Pmt1 activity on in vitro-transcribed tRNA^Glu and tRNA^Lys. In vitro-transcribed tRNAs (500 ng) were incubated with 3 µM Pmt1. Samples were analysed as in A. (E) Pmt1 activity on in vitro-transcribed tRNA^Asp and tRNA^Glu. In vitro-transcribed tRNA^Asp (500 ng) and tRNA^Glu (5 µg) were incubated with 3 µM Pmt1 or Pmt1C81A. Samples were analysed as in A. (F) Time course of tRNA^Asp methylation by Pmt1 (1.5 µM). The upper panel shows the time course of incorporation of radioactivity into the tRNA. The radioactive bands were analysed quantitatively and the data fitted to a single exponential reaction progress curve as shown in the lower panel.

Figure 2.

pmt1⁺ overexpression induces methylation of tRNA^Asp and tRNA^Glu in vivo in S. pombe. (A) In vivo RNA bisulfite sequencing of tRNA^Asp from total RNA of a pmt1Δ (AEP8) and a wt strain (AEP1) carrying an empty vector or a plasmid with pmt1⁺ expressed from the nmt1 promoter (nmt1pr-pmt1⁺, pAE1462). Cells were grown in supplemented EMM medium. The cytosine residues present in tRNA^Asp are indicated in the top row. Each subsequent row represents an independent clone that was sequenced. Black boxes indicated methylated cytosine, and gray or white boxes indicate unmethylated cytosines. The arrow indicates position C38 of tRNA^Asp. (B) In vivo methylation of tRNA^Glu on pmt1⁺ overexpression. Representation as in A. (C) Right two lanes, in vitro methylation of total RNA from a wt and a pmt1⁺ overexpressing strain (nmt1pr-pmt1⁺) by recombinant Pmt1. pmt1⁺ overexpression in vivo caused a reduced in vitro methylation signal. Left two lanes, methylation of in vitro transcribed tRNA^Asp and tRNA^AspC38A by Pmt1, representation as in Figure 1B.

Figure 3.

Regulation of Pmt1-dependent in vivo tRNA methylation by nutrient conditions. (A) RNA bisulfite sequencing of tRNA^Asp from wt (AEP1) and pmt1Δ (AEP8) cells cultured in S. pombe complete growth medium (YES) showed no in vivo methylation of tRNA^Asp at C38. (B) in vivo tRNA methylation was induced when cells were cultured in the presence of peptone. Total RNA of cells cultured in YES, YPD, YES with 2% peptone, or YPD in which the peptone had been omitted, was methylated in vitro using recombinant Pmt1 as in Figure 2C. (C) wt cells showed near 100% methylation of tRNA^Asp-C38 when cultured in S. cerevisiae complete growth medium (YPD), as measured by RNA bisulfite sequencing. Representation as in Figure 2A. (D) tRNA^Glu was not methylated at the C38 position in wt cells grown in YPD, as measured by RNA bisulfite sequencing. (E) Effect of nitrogen levels on Pmt1-dependent tRNA methylation. Cells were grown in ammonium-containing minimal medium (EMM, 0.5% ammonium chloride), 0.1% glutamate (EMMG) or with increased ammonium chloride (EMM + 2.5% NH₄Cl) before RNA extraction and methylation analysis as in Figure 2C.

Figure 4.

In vivo tRNA methylation depended on the kinase Sck2. (A) sck2Δ, but not sck1Δ or tor1Δ, caused a reduction of in vivo tRNA methylation. The indicated strains (AEP8, AEP1, AEP61, AEP62, AEP120) were grown in rich S. cerevisiae medium (YPD), and total RNA was methylated in vitro with recombinant Pmt1 as in Figure 2C. (B) sck2Δ, but not pka1Δ, caused a loss of in vivo tRNA methylation. Strains used were wt (AEP1), pmt1Δ (AEP8), pka1Δ (AEP117), pka1Δ pmt1Δ (AEP125), sck2Δ (AEP119) and sck2Δ pmt1Δ (AEP126). (C) In vivo tRNA methylation on pmt1⁺ overexpression required Sck2. Cells (AEP8, AEP125 or AEP126 transformed with pAE1429 or pAE1462) were grown in EMM medium for plasmid selection, and in vitro methylation by Pmt1 was performed as in Figure 2C.

Figure 5.

Trm4-dependent methylation of S. pombe tRNA^Asp. RNA bisulfite sequencing of tRNA^Asp from trm4aΔ (AEP102) and trm4bΔ (AEP103) cells cultured in S. pombe complete growth medium (YES) showed in vivo methylation of positions C48, C49, C60, C61 and C62. The double mutant trm4aΔ trm4bΔ (AEP162) showed a significant loss of m⁵C at these positions. Representation as in Figure 2A.