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Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA).

Müller S, Windhof IM, Maximov V, Jurkowski T, Jeltsch A, Förstner KU, Sharma CM, Gräf R, Nellen W - Nucleic Acids Res. (2013)

Bottom Line: Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress.However, dnmA expression only partially correlated with tRNA methylation in vivo.As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, Institute of Biochemistry, University Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany, Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg and Universität Potsdam, Institut für Biochemie und Biologie, Abt. Zellbiologie, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam - Golm.

ABSTRACT
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

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In vitro methylation of tRNAAsp(GUC). (A) In vitro methylation of tRNAAsp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl2 and by hDnmt2 at 5 mM Mg2+. The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3H-Me in the tRNAs. Reactions were run for the times indicated. (B) In vivo methylation of cytosines in tRNAAsp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNAAsp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. (C) In vitro methylation of small enriched RNA of a dnmAKO strain (ex vivo methylation). The methylation reaction was done for the times indicated.
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gkt634-F1: In vitro methylation of tRNAAsp(GUC). (A) In vitro methylation of tRNAAsp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl2 and by hDnmt2 at 5 mM Mg2+. The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3H-Me in the tRNAs. Reactions were run for the times indicated. (B) In vivo methylation of cytosines in tRNAAsp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNAAsp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. (C) In vitro methylation of small enriched RNA of a dnmAKO strain (ex vivo methylation). The methylation reaction was done for the times indicated.

Mentions: Previous experiments have shown that hDnmt2 can methylate a D. discoideum RNA in the size range of tRNAs in vitro (13). Here, we investigated whether the D. discoideum Dnmt2-homologue DnmA is also an active tRNAAsp methyltransferase. In vitro transcribed D. discoideum tRNAAsp(GUC) was used as a substrate for methylation by DnmA and hDnmt2 in vitro. The assays showed that the tRNA was efficiently methylated by both enzymes (Figure 1A). The reaction was time dependent, the activity was sensitive to Mg2+ concentrations and almost lost at 10 mM Mg2+. In contrast to the human enzyme, DnmA was inactive at 37°C (data not shown). Therefore, all reactions were carried out at 22°C where hDnmt2 still showed good activity.Figure 1.


Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA).

Müller S, Windhof IM, Maximov V, Jurkowski T, Jeltsch A, Förstner KU, Sharma CM, Gräf R, Nellen W - Nucleic Acids Res. (2013)

In vitro methylation of tRNAAsp(GUC). (A) In vitro methylation of tRNAAsp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl2 and by hDnmt2 at 5 mM Mg2+. The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3H-Me in the tRNAs. Reactions were run for the times indicated. (B) In vivo methylation of cytosines in tRNAAsp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNAAsp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. (C) In vitro methylation of small enriched RNA of a dnmAKO strain (ex vivo methylation). The methylation reaction was done for the times indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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gkt634-F1: In vitro methylation of tRNAAsp(GUC). (A) In vitro methylation of tRNAAsp(GUC) by DnmA at 2 mM, 5 mM and 10 mM MgCl2 and by hDnmt2 at 5 mM Mg2+. The upper panel shows ethidium bromide staining of the in vitro transcripts separated in a denaturing polyacrylamide gel, the lower panel shows incorporated 3H-Me in the tRNAs. Reactions were run for the times indicated. (B) In vivo methylation of cytosines in tRNAAsp(GUC) from different D. discoideum strains. Results of the RNA bisulfite sequencing (454 pyrosequencing) are given in percentage of reads. Numbers of sequence reads are shown in brackets. C49 is methylated by a different methyltransferase and thus serves as an internal standard. All 22 tRNAAsp(GUC) genes result in the same transcript, and no isoacceptors are encoded in the D. discoideum genome. (C) In vitro methylation of small enriched RNA of a dnmAKO strain (ex vivo methylation). The methylation reaction was done for the times indicated.
Mentions: Previous experiments have shown that hDnmt2 can methylate a D. discoideum RNA in the size range of tRNAs in vitro (13). Here, we investigated whether the D. discoideum Dnmt2-homologue DnmA is also an active tRNAAsp methyltransferase. In vitro transcribed D. discoideum tRNAAsp(GUC) was used as a substrate for methylation by DnmA and hDnmt2 in vitro. The assays showed that the tRNA was efficiently methylated by both enzymes (Figure 1A). The reaction was time dependent, the activity was sensitive to Mg2+ concentrations and almost lost at 10 mM Mg2+. In contrast to the human enzyme, DnmA was inactive at 37°C (data not shown). Therefore, all reactions were carried out at 22°C where hDnmt2 still showed good activity.Figure 1.

Bottom Line: Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress.However, dnmA expression only partially correlated with tRNA methylation in vivo.As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Kassel, Heinrich-Plett-Str. 40, 34132 Kassel, Germany, Institute of Biochemistry, University Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany, Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg and Universität Potsdam, Institut für Biochemie und Biologie, Abt. Zellbiologie, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam - Golm.

ABSTRACT
Although the DNA methyltransferase 2 family is highly conserved during evolution and recent reports suggested a dual specificity with stronger activity on transfer RNA (tRNA) than DNA substrates, the biological function is still obscure. We show that the Dictyostelium discoideum Dnmt2-homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNA(Asp(GUC)) in vitro and in vivo. By an ultraviolet-crosslinking and immunoprecipitation approach, we identified further DnmA targets. This revealed specific tRNA fragments bound by the enzyme and identified tRNA(Glu(CUC/UUC)) and tRNA(Gly(GCC)) as new but weaker substrates for both human Dnmt2 and DnmA in vitro but apparently not in vivo. Dnmt2 enzymes form transient covalent complexes with their substrates. The dynamics of complex formation and complex resolution reflect methylation efficiency in vitro. Quantitative PCR analyses revealed alterations in dnmA expression during development, cell cycle and in response to temperature stress. However, dnmA expression only partially correlated with tRNA methylation in vivo. Strikingly, dnmA expression in the laboratory strain AX2 was significantly lower than in the NC4 parent strain. As expression levels and binding of DnmA to a target in vivo are apparently not necessarily accompanied by methylation, we propose an additional biological function of DnmA apart from methylation.

Show MeSH
Related in: MedlinePlus