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. 2017 Sep 2;14(9):1241-1251.
doi: 10.1080/15476286.2016.1236170. Epub 2016 Nov 7.

The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA

Steffen Kaiser  1 , Tomasz P Jurkowski  2 , Stefanie Kellner  1 , Dirk Schneider  1 , Albert Jeltsch  2 , Mark Helm  1
Affiliations

The RNA methyltransferase Dnmt2 methylates DNA in the structural context of a tRNA

Steffen Kaiser et al. RNA Biol. .

Abstract

The amino acid sequence of Dnmt2 is very similar to the catalytic domains of bacterial and eukaryotic DNA-(cytosine 5)-methyltransferases, but it efficiently catalyzes tRNA methylation, while its DNA methyltransferase activity is the subject of controversial reports with rates varying between zero and very weak. By using composite nucleic acid molecules as substrates, we surprisingly found that DNA fragments, when presented as covalent DNA-RNA hybrids in the structural context of a tRNA, can be more efficiently methylated than the corresponding natural tRNA substrate. Furthermore, by stepwise development of tRNAAsp, we showed that this natural Dnmt2 substrate could be engineered to employ RNAs that act like guide RNAs in vitro. The 5'-half of tRNAAsp was able to efficiently guide methylation toward a single stranded tRNA fragment as would result from tRNA cleavage by tRNA specific nucleases. In a more artificial setting, a composite system of guide RNAs could ultimately be engineered to enable the enzyme to perform cytidine methylation on single stranded DNA in vitro.

Keywords: 5-methylcytosine; DNA; Dnmt2; RNA methylation; RNA modification; enzyme kinetics; modification pathway crosstalk; tRNA.

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Figures

Figure 1.
Figure 1.
Tritium incorporation assay suggests that Dnmt2 methylates a tRNA containing a deoxycytidine at position 38. (A) Cloverleaf structure of unmodified human cytosolic tRNAAsp. Position 38 has been engineered to contain rC, dC, or dm5C in three hybrid tRNAs of corresponding name. (B) In vitro methylation measured by tritium incorporation from 3H-SAM. Standard deviations of three replicates are given in Fig. S1.
Figure 2.
Figure 2.
LC-MS analysis confirms that Dnmt2 methylates a tRNA containing a deoxycytidine at position 38. (A) Fragmentation patterns and mass transitions used for scanning of rm5C and dm5C in LC-MS. (B) LC-MS analysis of all-ribo tRNAAsp and hybrid tRNAAspdC38 before and after in vitro methylation by human Dnmt2.
Figure 3.
Figure 3.
Dnmt2 methylates hybrid tRNAs containing up to 10 deoxynucleotides. (A) Positions of DNA substitutions in the tRNA cloverleaf in hybrid tRNAs. (B) Methylation of hybrid tRNAs of increasing DNA content. Average values and standard deviations are given in Fig. S1.
Figure 4.
Figure 4.
Relative methylation efficiency of covalent chimeric tRNAsAsp compared to the all-ribo tRNAAsp (entry 4c in Table 1), which is set to 100 on the black scale bar on the right.
Figure 5.
Figure 5.
Relative methylation efficiency of 2-component hybrid-chimeric tRNAsAsp compared to the all-ribo tRNAAsp (entry 4c/5b in Table 1) which is set to 100 on the black scale bar in the middle. Deviations from the native tRNAAsp sequence are presented in capital letters, DNA is highlighted in red.
Figure 6.
Figure 6.
Methylation of an RNA-guided DNA oligonucleotide by Dnmt2. (A) Structure of the hybridized construct. DNA shown in red. (B) Average values and standard deviations of 3 tritium incorporation assays are shown.
Figure 7.
Figure 7.
(A) Two-dimensional thin-layer chromatography of nucleosides on a 10 cm x 10 cm cellulose TLC plate. The starting point is marked by an X. (B) Methylation of an RNA-guided DNA oligonucleotide by Dnmt2. The oligonucleotides were hydrolyzed to nucleosides after the tritium incorporation assay, separated by 2D thin-layer chromatography and analyzed with the Cherenkov counter. Tritium could only be detected in m5dC. Average values and standard deviations of 3 experiments are shown. Control corresponds to background signal of the TLC plate. Note that an identical Fig. S4 with enhanced contrast can be found in the supplement.
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