title: Target recognition, RNA methylation activity and transcriptional regulation of the Dictyostelium discoideum Dnmt2-homologue (DnmA).
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.
authors.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.
authors.forename: Sara
authors.initials: S
authors.lastname: Müller
authors.forename: Indra M
authors.initials: IM
authors.lastname: Windhof
authors.forename: Vladimir
authors.initials: V
authors.lastname: Maximov
authors.forename: Tomasz
authors.initials: T
authors.lastname: Jurkowski
authors.forename: Albert
authors.initials: A
authors.lastname: Jeltsch
authors.forename: Konrad U
authors.initials: KU
authors.lastname: Förstner
authors.forename: Cynthia M
authors.initials: CM
authors.lastname: Sharma
authors.forename: Ralph
authors.initials: R
authors.lastname: Gräf
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Nellen
mesh_terms: D002453:Cell Cycle; D058639:Cold-Shock Response; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D005786:Gene Expression Regulation; D006801:Humans; D008745:Methylation; D015800:Protozoan Proteins / Q000235:genetics / Q000378:metabolism*; D012349:RNA, Transfer, Asp / Q000737:chemistry / Q000378:metabolism; D012352:RNA, Transfer, Glu / Q000378:metabolism; D012353:RNA, Transfer, Gly / Q000378:metabolism; D014158:Transcription, Genetic; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
chemical_list: D015800:Protozoan Proteins; D012349:RNA, Transfer, Asp; D012352:RNA, Transfer, Glu; D012353:RNA, Transfer, Gly; D012359:tRNA Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; C112218:TRDMT1 protein, human