Project description:Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. The absence of obvious mutant phenotypes under laboratory conditions suggested a function for Dnmt2 under non-physiological conditions. Indeed, Dnmt2 has recently been implicated in various aspects of the cellular stress response and the tRNA methyltransferase activity of Dnmt2 has been shown to interfere with stress-induced fragmentation of various tRNAs. We used adult animals and small RNA sequencing during a heat stress experiment to determine the tRNA fragment abundance and identities in wild-type and Dnmt2 mutant somatic tissues. Dnmt2 mutants produced tRNA fragments with different identities when compared to wild-type controls, indicating the accumulation of non-physiological tRNA-derived molecules in tissues without Dnmt2.
Project description:Mouse embryonic fibroblast (MEFs) cell lines and liver samples from Dnmt2 and Nsun2 single mutant, double mutant and wildtype mice were used to identify potential mRNA substrates of both proteins. MEFs were derived from day 13.5 embryos and immortalized by transfection with a plasmid expressing the SV40 large-T antigen. Total RNA of MEFs cell line at passage 9 and total RNA extracted from 2 months old mouse liver tissues of Dnmt2, Nsun2 single mutant, double mutant and wildtype mice were arrayed on Illumina MouseRef8 v2 chips.
Project description:Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. The absence of obvious mutant phenotypes under laboratory conditions suggested a function for Dnmt2 under non-physiological conditions. Indeed, Dnmt2 has recently been implicated in various aspects of the cellular stress response and the tRNA methyltransferase activity of Dnmt2 has been shown to interfere with stress-induced fragmentation of various tRNAs. We used adult animals and small RNA sequencing during a heat stress experiment to determine the tRNA fragment abundance and identities in wild-type and Dnmt2 mutant somatic tissues. Dnmt2 mutants produced tRNA fragments with different identities when compared to wild-type controls, indicating the accumulation of non-physiological tRNA-derived molecules in tissues without Dnmt2. 6 samples examined: heterozygous and Dnmt2 mutant under control, heat shock and recovery conditions
Project description:Mouse embryonic fibroblast (MEFs) cell lines and liver samples from Dnmt2 and Nsun2 single mutant, double mutant and wildtype mice were used to identify potential mRNA substrates of both proteins.
Project description:Dnmt2 and NSun2 are (cytosine-5) RNA methyltransferases. Using CRISPR/Cas9 we created null mutations in Dnmt2 and NSun2 genes in Drosophila melanogaster. We also ectopically expressed a wild type and catalytically inactive Dnmt2 form in the Dnmt2 mutant background. RNA bisulfite sequencing was used to follow RNA methylation at partical tRNA substrates.
Project description:Several organisms belonging to diverse animal groups have retained Dnmt2 as their only bona fide DNA methyltransferase gene. However, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which prompted us to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Using whole-genome bisulfite sequencing we show here that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in a Dnmt2-deficient fly strain. Furthermore, genetically modified mouse embryonic stem cells that had retained Dnmt2 as their only bona fide DNA methyltransferase gene, did not show any detectable DNA methylation patterns. Our results thus uncover fundamental differences among animal methylomes and suggest that Dnmt2-only organisms lack biologically relevant DNA methylation patterns. Whole methylome analysis of Mus musculus. One sample was analyzed containing DNA from Dnmt1-/-, Dnmt3a-/- and Dnmt3b-/- mice.