Cytosine methylation of tRNA-Asp by DNMT2 has a role in translation of proteins containing poly-Asp sequences

Abstract

The Dnmt2 RNA methyltransferase catalyses the methylation of C38 in the anticodon loop of tRNA-Asp, but the molecular role of this methylation is unknown. Here, we report that mouse aspartyl-tRNA synthetase shows a four to fivefold preference for C38-methylated tRNA-Asp. Consistently, a 30% reduced charging level of tRNA-Asp was observed in Dnmt2 knockout (KO) murine embryonic fibroblast cells. Gene expression analysis with fluorescent reporter proteins fused to an N-terminal poly-Asp sequence showed that protein synthesis of poly-Asp-tagged reporter proteins was reduced in Dnmt2 KO cells as well. The same effect was observed with endogenous proteins containing poly-Asp sequences, indicating that Dnmt2-mediated C38 methylation of tRNA-Asp regulates the translation of proteins containing poly-Asp sequences. Gene ontology searches for proteins containing poly-Asp sequences in the human proteome showed that a significant number of these proteins have roles in transcriptional regulation and gene expression. Hence, the Dnmt2-mediated methylation of tRNA-Asp exhibits a post-transcriptional regulatory role by controlling the synthesis of a group of target proteins containing poly-Asp sequences.

Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification

“…Studies on Dnmt2 were largely fuelled by the discovery of its tRNA methylation activity in 2006 [4], and studies of its role in translation are beginning to emerge [53,54]. The advent of ribosome profiling as a highly quantitative method for measuring translational effects opens up new avenues for the exploration of combined effects of Q and Dnmt2 on translation.…”

“…Since the major target for methylation by Dnmt2 is tRNA Asp , one study hypothesized that the absence of Dnmt2 would be particularly detrimental for the translation of proteins carrying a poly-aspartate stretch [53]. Indeed, reporter constructs with an N-terminal stretch of six consecutive aspartates was less efficiently expressed in mouse MEF cells lacking Dnmt2.…”

“…Indeed, reporter constructs with an N-terminal stretch of six consecutive aspartates was less efficiently expressed in mouse MEF cells lacking Dnmt2. This observation was extended to several native proteins containing poly-aspartate stretches, which showed lower levels in the absence of Dnmt2, indicating that Dnmt2-mediated tRNA methylation is required for efficient translation of proteins containing poly-aspartate sequences [53]. …”

“…In vitro, a 5.5-fold increase in Vmax/Km for the methylated tRNA as compared to the unmethylated substrate was observed [53]. Furthermore, this translated to a 30% lower level of charged tRNA Asp in vivo and indicated that the absence of m 5 C38 in cells leads to reduced availability of charged tRNA.…”

DNA Methylation Machinery in the Endometrium and Endometrial Cancer

“…Compared to other methyltransferases, DNMT2 is an RNA methyltransferase which can methylate cytosines in the anticodon loop of tRNA ASP despite its high sequence and structural similarity to other DNMT enzymes. This DNMT2-mediated methylation of tRNA ASP exhibits a post-transcriptional regulatory role in synthesis of proteins containing poly-Asp sequences (70).…”