Investigation of tRNA Modifications and their structural impact

DNA methylation at postion 5 of the cytosine appears in all 3 domains of life. The important role of these enzymes in evolutionary process is suggested by its the high conservation during evolution. Despite high sequence homology of Dnmt2 with bona fide DNA-methytransferases, only very weak DNA methyltransferase activity was reported. In contrast, it was shown by Goll et al. that Dnmt2 is able to methylate tRNA^Asp at cytosine 38. The aim of our work is the investigation and better understanding of the tRNA methylation and importance for epigenetic processes and the search of Dnmt2 targets other than tRNA.

We observed methylation levels of vitro transcribed tRNAs after incubation with human Dnmt2 in the presence of radioactively labeled cofactor S-Adenosyl-methionin, the methyl group donor. Methylation levels were assayed by measuring the incorporation of tritiated methyl groups into tRNA by liquid scintillation counting. Interestingly tRNA^Asp from Drosophila melanogaster was methylated by hDnmt2 whereas tRNA^Asp from Saccaromyces cerevisiae could not be efficiently modified. After comparison of their sequences we could determined a set of nucleotides which seems to be necessary for the recognition of tRNA^Asp by Dnmt2.