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

Sara M uller 2 Indra M. Windhof 2 Vladimir Maximov 2 Tomasz Jurkowski 1 Albert Jeltsch 1 Konrad U. F orstner 0 Cynthia M. Sharma 0 Ralph Gr af 3 Wolfgang Nellen 2 0 Research Center for Infectious Diseases (ZINF), University of W urzburg , Josef-Schneider-Str. 2/Bau D15, 97080 W urzburg 1 Institute of Biochemistry, University Stuttgart , Pfaffenwaldring 55, 70569 Stuttgart, Germany 2 Department of Genetics, University of Kassel , Heinrich-Plett-Str. 40, 34132 Kassel, Germany 3 Universita t Potsdam, Institut fu r Biochemie und Biologie , Abt. Zellbiologie, Karl-Liebknecht-Strasse 24-25, 14476 Potsdam - Golm 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 Dnmt2homologue DnmA is an active tRNA methyltransferase that modifies C38 in tRNAAsp(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 tRNAGlu(CUC/UUC) and tRNAGly(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. - Dnmt2 is a member of the eukaryotic DNA methyltransferase family. A few model organisms, especially Drosophila melanogaster, Schizosaccharomyces pombe, Entamoeba histolytica and Dictyostelium discoideum contain only one Dnmt2 homologue but lack the more active homologues Dnmt1 and Dnmt3. Though highly conserved during evolution, loss of Dnmt2 homologues has no obvious phenotypic effects in Mus musculus (1), D. melanogaster (2), S. pombe (3) and D. discoideum (4,5). In Danio rerio, disruption of dnmt2 has been reported to cause pleiotropic effects (6) and in E. histolytica a gene disruption could not be obtained, suggesting that Dnmt2 was required for viability (7). On closer inspection, more subtle long-term effects of Dnmt2 loss were observed: in D. melanogaster, for example, H4K20me3 was strongly reduced, and telomeric sequences were lost (2). Durdevic et al. (8) recently showed that in D. melanogaster, Dnmt2 was also involved in virus control. At least in D. melanogaster, E. histolytica and D. discoideum, low amounts of DNA methylation, especially on retroelements, were detected and attributed to Dnmt2, although these data are controversially discussed (911). In 2006, Bestors group reported that Dnmt2 rather methylates transfer RNA (tRNA) than DNA in M. musculus, D. melanogaster and Arabidopsis thaliana. Specifically, they found C38 in tRNAAsp as the major or only substrate for the enzyme (1). A dual role of Dnmt2 had been discussed (12), and Jurkowski et al. (13) could show that tRNAAsp was methylated by an enzymatic mechanism characteristic for DNA methyltransferases rather than by the reaction pathways of enzymes that methylate RNAs. tRNAAsp methylation activity was also reported for the Dnmt2-homologues from E. histolytica (14) and D. melanogaster (15). Schaefer et al. (15) showed that D. melanogaster tRNAVal(CAC) and tRNAGly(GCC) were also methylated at position C38 in vivo and by human Dnmt2 (hDnmt2) in vitro. They further found that dnmt2 knockout flies were more sensitive to oxidative stress. A more detailed analysis of tRNAAsp(GUC) and tRNAGly(GCC) suggested that methylation protected tRNAs from stress-induced cleavage (15). Doubleknockout mutant mice of Dnmt2 and Nsun2, the second known m5C-tRNA-methyltransferase in higher eukaryotes, showed a phenotype with impaired cellular differentiation, an overall reduction in protein synthesis and early lethality (16). Recently, we identified tRNAGlu(UUC) as an additional novel substrate of Pmt1, the Dnmt2-homologue in S. pombe (17). Pmt1-dependent tRNA methylation seemed to be regulated by nutrient conditions. Nutritional control was also reported for Ehmeth, the Dnmt2 homologue from E. histolytica that is inhibited by the glycolytic enzyme enolase (14,18). Here, we demonstrate that recombinant D. discoideum DnmA and hDnmt2 can methylate tRNAAsp(GUC), tRNAGlu(UUC) and tRNAGly(GCC) from D. discoideum in vitro with different efficiencies. Both enzymes formed covalent complexes [22] with specific tRNAs with similar kinetics, but they were significantly slower for the minor substrate tRNAGlu than for the major substrate tRNAAsp. Ultraviolet (UV)-crosslinking and immunoprecipitation (CLIP) experiments showed that specific fragments of the three target tRNAs associated with DnmA in vivo. However, in vivo, only methylation of tRNAAsp(GUC) by DnmA was confirmed, whereas methylation of the target nucleotide C38 in other substrates was not detectable by bisulfite sequencing. As revealed by quantitative reverse transcription (RT)PCR, dnmA is differentially expressed in development, cell cycle and in the recovery phase after temperature stress. The increase in dnmA expression levels correlated with elevated tRNAAsp methylation in development but not after temperature stress. The other targets identified by in vitro methylation and by CLIP were apparently also not methylated in vivo in development. Our data document that additional RNA molecules can serve as substrates for Dnmt2 binding and that the full range of targets is probably not yet recognized. The results also suggest that binding of the methyltransferase to an RNA molecule not necessarily results in methylation but may have different biological functions. MATERIALS AND METHODS Dictyostelium discoideum cultures and nomenclature Dictyostelium discoideum AX2-214 was grown in HL5+ medium (ForMedium) containing 50 mg/ml Ampicillin, 0.25 mg/ml Amphotericin-B, 100 mg/ml Penicillin/ Streptomycin (PAA) at 22 C, constant light under selective conditions as required. NC4 cells were grown in a suspension of Klebsiella aerogenes in phosphate buffer. When indicated, AX2 cells were also grown in bacterial suspension to allow for comparison with NC4. For cold treatment cells (1 106/ml) were shaken at 4 C for 214 h. Cells were allowed to recover at 22 C for 2 h before RNA isolation. For synchronization, cells at a density of 13 105/ml were incubated at 4 C over night. Before cold treatment, cells were briefly cooled down in a water bath with ice. For synchronization, cells were then warmed up in a 25 C water bath before cultivating at 22 C. Synchronization was measured by counting cells every 30 mi (...truncated)