RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

Abstract

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.

Figure 1.

Dnmt2 is a multisubstrate tRNA methyltransferase. (A) Quantitative PCR analysis of Dnmt2 mRNA expression in Drosophila tissues. Expression was normalized to rp49 levels. (B) Identification of novel Dnmt2 substrate tRNAs. Deep bisulfite sequencing analysis of various tRNAs in wild-type (control) and Dnmt2 mutant (Dnmt2 mut.) flies. Numbers of sequence reads are indicated at the top of each panel. Diagrams show fractions of nondeaminated cytosines, thus revealing the pattern of m5C modification in individual tRNAs. Cytosines that were found to be methylated in a Dnmt2-dependent manner are C38 in tRNA^Asp-GTC, tRNA^Val-AAC, and tRNA^Gly-GCC (highlighted by bold numbers).

Figure 2.

Loss of Dnmt2 causes increased stress sensitivity. (A) Viability plot of Dnmt2⁹⁹ mutant, control (Dnmt2^rev), and transgenic rescue (Dnmt2^genTG) flies reared at elevated temperature (29°C). For each genotype and sex, the survival of 20 flies (3 d old) was counted over a period of 39 d. Error bars represent standard deviations from five independent experiments. (B) Dnmt2 mutant flies show elevated sensitivity to paraquat. For each genotype, 20 male or female adult flies were exposed to paraquat, and live flies were counted for 5 d. Error bars represent standard deviations from five independent experiments. (C) Dnmt2 mutant flies show elevated sensitivity to H₂O₂. Twenty male or female adult flies were exposed to 1% H₂O_2, and live flies were counted for 3 d. Error bars represent standard deviations from five independent experiments.

Figure 3.

Dnmt2 is associated with stress compartments. (A) Immunofluorescence analysis of FMR1 in S2 cells expressing Dnmt2-EGFP. FMR1 localizes to the cytoplasm, whereas Dnmt2-EGFP is localized ubiquitiously to the cytoplasm and nucleus in control cells. Upon heat shock (1 h at 40°C), FMR1 concentrates in large cytoplasmic granules where the signal colocalizes with granular Dnmt2-EGFP. (B) Similar observations were made in ovaries after heat shock. (C) Immunofluorescence analysis of the P-body-associated protein ME31B on fixed Dnmt2-EGFP ovaries. After heat shock, the number of ME31B foci increased, and colocalization with Dnmt2-EGFP structures could be observed. Bars: A, 10 μM; B,C, 1 μM.

Figure 4.

Stress-induced cleavage of Dnmt2 substrate tRNAs. (A) Northern analysis of tRNA^Gly-GCC cleavage in S2 cells that were heat-shocked (1 h at 40°C) for the times (in minutes) indicated. 5.8S rRNA was used as a loading control. (B) Cleavage of tRNA^Gly-GCC in S2 cells that were treated with H₂O₂ (1 mM H₂O₂, 60 min) or sodium arsenite (0.5 mM SA, 90 min). (C) Cleavage of tRNA^Gly-GCC in wild-type (D2^+/+) or Dnmt2 mutant (D2^−/−) ovaries (resected 35 d after hatching) that were heat-shocked (+) in medium at 40°C. Controls (−) show results from parallel experiments with ovary incubation at 25°C. (D) Northern analysis of tRNA^Gly-GCC and tRNA^Asp-GTC cleavage in S2 cells that allow inducible overexpression of Dnmt2 (iD2). Full-length tRNAs are marked by gray arrows, and tRNA fragments are marked by black arrowheads.

Figure 5.

Dnmt2-mediated tRNA methylation inhibits endonucleolytic cleavage by angiogenin. (A) Dnmt2 overexpression (ecD2) inhibits angiogenin-induced tRNA cleavage. tRNA cleavage was induced in S2 cells by the addition of recombinant angiogenin (rANG) to the medium (1 μg/mL), and was analyzed by Northern blotting. Full-length tRNAs (gray arrows) and cleavage products (black arrowheads) are shown from the same original blot. (B) Dnmt2 substrate tRNAs isolated from wild-type embryos (D2^+/+) are protected against angiogenin cleavage in vitro. No protection effect was observed with Dnmt2 substrate tRNAs isolated from Dnmt2 mutant embryos (D2^−/−), or with tRNA^Met-ATG, which is not a Dnmt2 substrate. (C) Dnmt2-mediated protection against angiogenin cleavage is conserved in mice. tRNA was purified from wild-type (D2^+/+) or Dnmt2 mutant (D2^−/−) MEFs, incubated with recombinant angiogenin (1 μM, 60 min), and analyzed by Northern blotting. Full-length tRNAs are marked by gray arrows, and tRNA fragments are marked by black arrowheads.