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Abstract

Dnmt2 genes encode highly conserved and widely distributed RNA methyltransferases with poorly understood biological functions. Recently, it has been shown that Dnmt2-mediated tRNA methylation interfered with stress-induced tRNA fragmentation, which suggested roles for Dnmt2 during cellular stress responses. The aim of this work was to investigate the roles of Dnmt2 during the response to biotic (pathogens), abiotic (heat) and endogenous RNA (transposon) stress in an attempt to further characterize the biological function of Dnmt2. Using the model organism Drosophila melanogaster and molecular and biochemical approaches this investigation revealed important roles for Dnmt2 in the control of exogenous and endogenous mobile elements. Dnmt2 mutants showed stress- and age-dependent morphological phenotypes. Molecular analysis identified increased levels of RNA viruses in Dnmt2 mutants and virus infection experiments confirmed that Dnmt2 mutants failed to control specific pathogens like Drosophila C virus (DCV). Expression analysis for immune response genes showed that Dnmt2 mutant flies were not able to efficiently activate innate immune responses. Importantly, the RNA methyltransferase function of Dnmt2 contributed to virus control and binding of Dnmt2-containing protein complexes to DCV-derived RNA implicated RNA methylation in the process. These findings revealed a novel biological role for Dnmt2 enzymes and suggest that RNA methylation may regulate viral RNA metabolism. To extend the investigation of Dnmt2 function in mobile element control to endogenous sequences, transposon control in Dnmt2 mutants was investigated. Dnmt2 mutants showed defects in the re-silencing of heat shock-induced transposons and this depended on the catalytic activity of Dnmt2. Furthermore, increased transposon-derived DNA and transposon mobility could be detected in Dnmt2 mutants. Heat shock caused significantly different tRNA fragmentation patterns in Dnmt2 mutants and Dnmt2 substrate tRNA fragments associated with Argonaute-2 after heat shock. Further analysis showed that Dnmt2 mutants accumulated increasing levels of Dicer-2-dependent double-stranded RNA precursors leading to inefficient endo-siRNA production. Consequently, Dnmt2 mutants displayed signs of mis-regulated siRNA pathways including reduced transposon silencing, which led to genomic instability. These findings uncovered a stress-related function for Dnmt2 in transposon regulation, which suggests that the increase in stress-induced tRNA fragments in Dnmt2 mutants affects the efficiency of small RNAi pathways. This thesis describes novel functions of Dnmt2 in cellular stress responses to biotic and abiotic stresses and indicates that the high conservation of Dnmt2 enzymes is founded in its contribution to defense mechanisms against external and internal mobile elements.