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. 2014 Jun;42(10):6487-96.
doi: 10.1093/nar/gku256. Epub 2014 Apr 7.

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu

Raghuvaran Shanmugam  1 , Muktak Aklujkar  2 , Matthias Schäfer  3 , Richard Reinhardt  4 , Olaf Nickel  5 , Gunter Reuter  5 , Derek R Lovley  2 , Ann Ehrenhofer-Murray  6 , Wolfgang Nellen  7 , Serge Ankri  8 , Mark Helm  9 , Tomasz P Jurkowski  1 , Albert Jeltsch  10
Affiliations

The Dnmt2 RNA methyltransferase homolog of Geobacter sulfurreducens specifically methylates tRNA-Glu

Raghuvaran Shanmugam et al. Nucleic Acids Res. 2014 Jun.

Abstract

Dnmt2 enzymes are conserved in eukaryotes, where they methylate C38 of tRNA-Asp with high activity. Here, the activity of one of the very few prokaryotic Dnmt2 homologs from Geobacter species (GsDnmt2) was investigated. GsDnmt2 was observed to methylate tRNA-Asp from flies and mice. Unexpectedly, it had only a weak activity toward its matching Geobacter tRNA-Asp, but methylated Geobacter tRNA-Glu with good activity. In agreement with this result, we show that tRNA-Glu is methylated in Geobacter while the methylation is absent in tRNA-Asp. The activities of Dnmt2 enzymes from Homo sapiens, Drosophila melanogaster, Schizosaccharomyces pombe and Dictyostelium discoideum for methylation of the Geobacter tRNA-Asp and tRNA-Glu were determined showing that all these Dnmt2s preferentially methylate tRNA-Asp. Hence, the GsDnmt2 enzyme has a swapped transfer ribonucleic acid (tRNA) specificity. By comparing the different tRNAs, a characteristic sequence pattern was identified in the variable loop of all preferred tRNA substrates. An exchange of two nucleotides in the variable loop of murine tRNA-Asp converted it to the corresponding variable loop of tRNA-Glu and led to a strong reduction of GsDnmt2 activity. Interestingly, the same loss of activity was observed with human DNMT2, indicating that the variable loop functions as a specificity determinant in tRNA recognition of Dnmt2 enzymes.

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Figures

Figure 1.
Figure 1.
G. sulfurreducens Dnmt2 methylates C38 of tRNAAsp. (A) Methylation of D. melanogaster tRNAAsp and murine-tRNAAsp with the GsDnmt2 enzyme. Control reactions using the catalytically inactive GsDnmt2 mutant C74A and Dm-tRNAAsp C38U mutant show no methylation. (B) Methylation of tRNAAsp from G. sulfurreducens with GsDnmt2 showing weak methylation. Control reactions with the C38U mutant of the tRNAAsp or the catalytically inactive C74A variant of GsDnmt2 showed no methylation signal. (C) Summary of the tRNA methylation data reporting average methylation rates and SEM taken from two to three independent experiments.
Figure 2.
Figure 2.
GsDnmt2 prefers methylation of G. sulfurreducens tRNAGlu over tRNAAsp. (A) Examples of in vitro methylation of Geobacter tRNAAsp and tRNAGlu by GsDnmt2. (B) Average methylation rates and SEM based on three repeats of the experiments. Panel (B) also includes results of control reactions with tRNAAsp and tRNAGlu C38U variants and with the catalytically inactive C74A GsDnmt2 protein. The error bars indicate the SEM from at least three different experimental repeats.
Figure 3.
Figure 3.
tRNAGlu but not tRNAAsp is methylated at C38 in G. sulfurreducens. The figure shows results of RNA bisulfite sequencing of tRNAAsp, tRNAGlu, tRNAGlu-2 and tRNAVal from total RNA isolated from G. sulfurreducens. The cytosine residues present in the corresponding tRNA are indicated in the top row. Each subsequent row represents an independent clone that was sequenced. Black boxes indicate methylated cytosines and white boxes indicate unmethylated cytosines. The arrows highlight the C38 positions.
Figure 4.
Figure 4.
Dnmt2 enzymes from various species prefer methylation of G. sulfurreducens tRNAAsp. (A) Examples of methylation kinetics are shown for wild-type Dnmt2 enzymes from S. pombe (Pmt1), D. discoideum (DnmA), D. melanogaster and H. sapiens using in vitro transcribed Gs-tRNAAsp and Gs-tRNAGlu and their respective C38U variants. (B) Average methylation rates and SEM based on two repeats of the experiments.
Figure 5.
Figure 5.
Comparison of the specificities of Dnmt2 homologs from different species for methylation of Geobacter tRNAAsp and tRNAGlu. Data are replotted from Figures 2B and 4B and the relative activities of the Asp/Glu pairs were always normalized to the more active substrate for a better comparison. Error bars were based on normalized individual experiments and correspond to the SEM of two to three repeats.
Figure 6.
Figure 6.
Clover leaf structure of the tRNAAsp and tRNAGlu from different species. The target C38 is printed in bold and highlighted by a gray circle for the preferred substrate tRNA and by a white circle for the non-preferred one. Bases in the variable loop are colored. The sequences of human tRNAAsp and tRNAGlu are identical to that of murine-tRNAAsp and tRNAGlu.
Figure 7.
Figure 7.
Methylation of tRNA variable loop mutants by GsDnmt2 and human DNMT2. (A) Methylation of murine-tRNAAsp with its original variable loop or with the variable loop of murine-tRNAGlu by Geobacter Dnmt2 (left pair of bars) or human DNMT2 (right pair of bars). (B) GsDnmt2 methylation of Gs-tRNAGlu with its original variable loop or with variable loops from Gs-tRNAAsp or murine-tRNAGlu. The figure shows averages of the methylation rates normalized to the wild-type tRNA. Error bars indicate SEM derived from two repeats of the experiments.
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References

    1. Helm M. Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res. 2006;34:721–733. - PMC - PubMed
    1. Carell T., Brandmayr C., Hienzsch A., Muller M., Pearson D., Reiter V., Thoma I., Thumbs P., Wagner M. Structure and function of noncanonical nucleobases. Angew. Chem. Int. Ed. Engl. 2012;51:7110–7131. - PubMed
    1. Machnicka M.A., Milanowska K., Osman Oglou O., Purta E., Kurkowska M., Olchowik A., Januszewski W., Kalinowski S., Dunin-Horkawicz S., Rother K.M., et al. MODOMICS: a database of RNA modification pathways–2013 update. Nucleic Acids Res. 2013;41:D262–D267. - PMC - PubMed
    1. El Yacoubi B., Bailly M., de Crecy-Lagard V. Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu. Rev. Genet. 2012;46:69–95. - PubMed
    1. Jeltsch A. Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem. 2002;3:274–293. - PubMed

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