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Abstract

Dnmt2 is a highly conserved RNA methyltransferase that is responsible for cytosine-C5 methylation of C38 in the anticodon-loop of tRNAAspGUC, tRNAValAAC, and tRNAGlyGCC. This modification has been shown to contribute to tRNA integrity and function. Additionally, it was demonstrated that RNA-mediated inheritance in mice depends on the presence of Dnmt2. Since the mechanistic role of Dnmt2 in many biological processes remains elusive, the aim of this thesis was to further functionally characterize Dnmt2 in the mouse. A special focus was set on tissues and cells which had a published Dnmt2-/- phenotype. Dnmt2 characterization thus concentrated on tissues and cells involved in inheritance, i.e. sperm as the transmitting cells and early embryos as the target. Phenotypic characterization showed that relevant Dnmt2-/- tissues were morphologically indistinguishable from those of wild type mice. Sperm, testis, and embryonic tissues showed proper differentiation and tissue characteristics. However, high Dnmt2 expression in testis and sperm supported its role in inheritance. Due to Dnmt2’s known role in RNA methylation, RNAs were analysed in depth in the relevant tissues. RNA-Seq experiments illustrated that RNA expression levels in sperm, testis, and embryonic samples showed only minor differences between wild type and Dnmt2-/-. For RNAs that were further investigated, the potential differences did not appear to have an immediate effect on processes associated with these RNAs. Importantly, targeted bisulfite sequencing and Northern blots showed that Dnmt2 is responsible for tRNA methylation and stability in the male germline. Furthermore, a whole-transcriptome bisulfite sequencing approach confirmed that Dnmt2 specifically methylates tRNAAspGUC, tRNAValAAC, and tRNAGlyGCC, and most likely no other RNAs. The methylation and assurance of integrity of these tRNAs is thus suspected to play a pivotal role in RNA-mediated inheritance. For further functional characterization of Dnmt2, a screen of different physiological as well as non-physiological conditions in cell culture and live mice was carried out to define factors that regulate Dnmt2 activity. It was found that depriving cell cultures and live mice of the micronutrient queuine decreases C38 methylation of tRNAAspGUC. In conclusion, this thesis shows that Dnmt2 is a highly specific tRNA methyltransferase, which confers stability to tRNAs in sperm, and responds to environmental stimuli in mice.