Poster

Dnmt2: A multi-substrate RNA methyltransferase?

Sara Müller¹, Vladimir Maximov², Susanne Junk³, Cynthia M. Sharma⁴, Jörg Vogel⁵, Wolfgang Nellen⁶

1 Dept. of Genetics, University of Kassel, Heinrich Plett-Str. 40, 34132 Kassel

2 Dept. of Genetics, University of Kassel, Heinrich Plett-Str. 40, 34132 Kassel

3 Dept. of Genetics, University of Kassel, Heinrich Plett-Str. 40, 34132 Kassel

4 Institute for Molecular Infection Biology, Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg

5 Institute for Molecular Infection Biology, Research Center for Infectious Diseases (ZINF), University of Würzburg, Josef-Schneider-Str. 2/Bau D15, 97080 Würzburg

6 Dept. of Genetics, University of Kassel, Heinrich Plett-Str. 40, 34132 Kassel

Abstract

The DNA methyltransferase 2 (Dnmt2) is the most conserved DNA methyltransferase throughout all kingdoms of life; however, its substrate specificity and biological function are still enigmatic.

Although weak m⁵C-DNA methylation on retroelements has been reported for “Dnmt2-only” organisms like Dictyostelium, Drosophila and Entamoeba, its role is still controversially discussed [1-5]. Recent data have shown methyltransferase activity on tRNA in vitro and in vivo for different Dnmt2 homologues and, thus, suggested a dual activity [6, 7].

Moreover, in vitro methylation analysis of the Dictyostelium Dnmt2-homologue, DnmA, has also revealed a tRNA methylation activity with a high specificity for tRNA^Asp and a reduced one for tRNA^Glu. However, the overall methylation activity seems to be weaker for recombinant DnmA than for recombinant human Dnmt2 (hDnmt2).

Initial electromobility shift assays indicate no differences in binding specificity of DnmA between sense and antisense tRNA transcripts. In contrast, covalent complexes which appear only during the methylation process can exclusively be observed with sense tRNA transcripts. Interestingly, covalent complexes are also formed with DNA oligos but without showing significant turnover.

To identify further RNA-substrates, a co-immunoprecipitation of GFP-tagged DnmA was combined with454 sequencing of associated RNAs [8]. This approach revealed several additional DnmA candidate substrates. In addition, rnu2 (U2 snRNA) but not sno7 (snoRNA 7) and two previously not annotated intergenic transcripts could be methylated to a low extend in vitro by DnmA and Dnmt2 in a first validation experiment. In summary, our results thus indicate that DnmA has additional substrates and roles in RNA methylation than previously anticipated.

References

1. Kuhlmann, M., et al., Silencing of retrotransposons in Dictyostelium by DNA methylation and RNAi. Nucleic Acids Res, 2005. 33(19): p. 6405-17.

2. Kunert, N., et al., A Dnmt2-like protein mediates DNA methylation in Drosophila. Development, 2003. 130(21): p. 5083-90.

3. Phalke, S., et al., Retrotransposon silencing and telomere integrity in somatic cells of Drosophila depends on the cytosine-5 methyltransferase DNMT2. Nat Genet, 2009. 41(6): p. 696-702.

4. Schaefer, M., et al., Lack of evidence for DNA methylation of Invader4 retroelements in Drosophila and implications for Dnmt2-mediated epigenetic regulation. Nat Genet. 42(11): p. 920-1; author reply 921.

5. Banerjee, S., et al., Entamoeba histolytica DNA methyltransferase (Ehmeth) is a nuclear matrix protein that binds EhMRS2, a DNA that includes a scaffold/matrix attachment region (S/MAR). Mol Biochem Parasitol, 2005. 139(1): p. 91-7.

6. Goll, M.G., et al., Methylation of tRNAAsp by the DNA methyltransferase homolog Dnmt2. Science, 2006. 311(5759): p. 395-8.

7. Jeltsch, A., et al., Two substrates are better than one: dual specificities for Dnmt2 methyltransferases. Trends Biochem Sci, 2006. 31(6): p. 306-8.

8. Sittka, A., et al., Deep sequencing analysis of small noncoding RNA and mRNA targets of the global post-transcriptional regulator, Hfq. PLoS Genet, 2008. 4(8): p. e1000163.

DOI^®: 10.3288/contoo.paper.1124