Determinants of DNMT2/TRDMT1 preference for substrates tRNA and DNA during the evolution.

RNA methyltransferase DNMT2/TRDMT1 is the most conserved member of the DNMT family from bacteria to plants and mammals. In previous studies, we found some determinants for tRNA recognition of DNMT2/TRDMT1, but the preference mechanism of this enzyme for substrates tRNA and DNA remains to be explored. In the present study, CFT-containing target recognition domain (TRD) and target recognition extension domain (TRED) in DNMT2/TRDMT1 play a crucial role in the substrate DNA and RNA selection during the evolution. Moreover, the classical substrate tRNA for DNMT2/TRDMT1 had a characteristic sequence CUXXCAC in the anticodon loop. Position 35 was occupied by U, making cytosine-38 (C38) twist into the loop, whereas C, G or A was located at position 35, keeping the C38-flipping state. Hence, the substrate preference could be modulated by the easily flipped state of target cytosine in tRNA, as well as TRD and TRED. Additionally, DNMT2/TRDMT1 cancer mutant activity was collectively mediated by five enzymatic characteristics, which might impact gene expressions. Importantly, G155C, G155V and G155S mutations reduced enzymatic activities and showed significant associations with diseases using seven prediction methods. Altogether, these findings will assist in illustrating the substrate preference mechanism of DNMT2/TRDMT1 and provide a promising therapeutic strategy for cancer.

Restricted tRNA methylation by intermolecular disulfide bonds in DNMT2/TRDMT1.

Reportedly, DNMT2/TRDMT1 mainly methylates tRNAs at C38 and prevents them from the cleavage under stress. It also plays an essential role in the survival and physiological homeostasis of organisms. Nevertheless, DNMT2/TRDMT1 exhibits much weaker tRNA methylation activity in vitro than other tRNA methyltransferases, TrmD and Trm5. Here, we explored the restricted tRNA methylation mechanism by DNMT2/TRDMT1. In the current study, the optimized buffer C at 37 °C was the best condition for DNMT2/TRDMT1 activation. Of note, Dithiothreitol (DTT) was an indispensable component for this enzyme catalysis. Moreover, reductants took similar effects on the conformation change and oligomeric formation of DNMT2/TRDMT1. Ultimately, LC-MS/MS result revealed that C292-C292 and C292-C287 were predominant intermolecular disulfide bonds in recombinant DNMT2/TRDMT1. Notably, DNMT2/TRDMT1 existed primarily as dimers via intermolecular disulfide bonds C79-C24, C292-C292, and C222-C24 in HEK293T cells. GSSG stress enhanced tRNA methylation level in the early stage of stress, whereas the DNMT2/TRDMT1 activity might be unfavorable along with this enzyme accumulation in the nucleus. Excitingly, GSH stress downregulated the DNMT2/TRDMT1 expression and promoted tRNA methylation in cells, probably through breaking intermolecular disulfide bonds in this enzyme. Thus, our findings demonstrated restricted tRNA methylation by disulfide bonds in DNMT2/TRDMT1, and will provide important implications for redox stress related-diseases.