The function of cytosine-C5 methylation, a widespread modification of tRNAs, has remained obscure, particularly in mammals. We have now developed a mouse strain defective in cytosine-C5 tRNA methylation, by disrupting both the Dnmt2 and the NSun2 tRNA methyltransferases. Although the lack of either enzyme alone has no detectable effects on mouse viability, double mutants showed a synthetic lethal interaction, with an underdeveloped phenotype and impaired cellular differentiation. tRNA methylation analysis of the double-knockout mice demonstrated complementary target-site specificities for Dnmt2 and NSun2 and a complete loss of cytosine-C5 tRNA methylation. Steady-state levels of unmethylated tRNAs were substantially reduced, and loss of Dnmt2 and NSun2 was further associated with reduced rates of overall protein synthesis. These results establish a biologically important function for cytosine-C5 tRNA methylation in mammals and suggest that this modification promotes mouse development by supporting protein synthesis.

Summary The discovery of RNAs (e.g. mRNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function in delivering additional paternal information aside from solely providing the DNA1. Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2, 3 and metabolic disorders4–6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat diet (HFD)-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30–40nt RNA fractions that are induced by HFD. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs (tsRNAs) and rRNA-derived small RNAs (rsRNA-28S), which might be essential in composing a sperm RNA ‘coding signature’ that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.

The Dnmt2 enzyme utilizes the catalytic mechanism of eukaryotic DNA methyltransferases to methylate several tRNAs at cytosine 38. Dnmt2 mutant mice, flies, and plants were reported to be viable and fertile, and the biological function of Dnmt2 has remained elusive. Here, we show that endochondral ossification is delayed in newborn Dnmt2-deficient mice, which is accompanied by a reduction of the haematopoietic stem and progenitor cell population and a cell-autonomous defect in their differentiation. RNA bisulfite sequencing revealed that Dnmt2 methylates C38 of tRNA AspGTC, GlyGCC, and ValAAC, thus preventing tRNA fragmentation. Proteomic analyses from primary bone marrow cells uncovered systematic differences in protein expression that are due to specific codon mistranslation by tRNAs lacking Dnmt2-dependent methylation. Our observations demonstrate that Dnmt2 plays an important role in haematopoiesis and define a novel function of C38 tRNA methylation in the discrimination of near-cognate codons, thereby ensuring accurate polypeptide synthesis.

Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing.Using RNAs from mouse embryonic stem cells we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA.In addition, mRNAs were found to be very sparsely methylated or not methylated at all.Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q‐tRNA levels promote Dnmt2‐mediated methylation of tRNA Asp and control translational speed of Q‐decoded codons as well as at near‐cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ‐free mice fed with a queuosine‐deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.

RNA–mediated transmission of phenotypes is an important way to explain non-Mendelian heredity. We have previously shown that small non-coding RNAs can induce hereditary epigenetic variations in mice and act as the transgenerational signalling molecules. Two prominent examples for these paramutations include the epigenetic modulation of the Kit gene, resulting in altered fur coloration, and the modulation of the Sox9 gene, resulting in an overgrowth phenotype. We now report that expression of the Dnmt2 RNA methyltransferase is required for the establishment and hereditary maintenance of both paramutations. Our data show that the Kit paramutant phenotype was not transmitted to the progeny of Dnmt2−/− mice and that the Sox9 paramutation was also not established in Dnmt2−/− embryos. Similarly, RNA from Dnmt2-negative Kit heterozygotes did not induce the paramutant phenotype when microinjected into Dnmt2-deficient fertilized eggs and microinjection of the miR-124 microRNA failed to induce the characteristic giant phenotype. In agreement with an RNA–mediated mechanism of inheritance, no change was observed in the DNA methylation profiles of the Kit locus between the wild-type and paramutant mice. RNA bisulfite sequencing confirmed Dnmt2-dependent tRNA methylation in mouse sperm and also indicated Dnmt2-dependent cytosine methylation in Kit RNA in paramutant embryos. Together, these findings uncover a novel function of Dnmt2 in RNA–mediated epigenetic heredity.

Cytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here, we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associated with bisulfite sequencing. By using RNAs from mouse embryonic stem cells, we performed a comprehensive methylation analysis of mouse tRNAs, rRNAs, and mRNAs. Our approach identified all known methylation marks in tRNA and two previously unknown but evolutionary conserved marks in 28S rRNA. In addition, mRNAs were found to be very sparsely methylated or not methylated at all. Finally, the tRNA-specific activity of the DNMT2 methyltransferase could be resolved at single-base resolution, which provided important further validation. Our approach can be used to profile cytosine-5 RNA methylation patterns in many experimental contexts and will be important for understanding the function of cytosine-5 RNA methylation in RNA biology and in human disease.

Genomic concepts are based on the assumption that phenotypes arise from the expression of genetic variants. However, the presence of non-Mendelian inheritance patterns provides a direct challenge to this view and suggests an important role for alternative mechanisms of gene regulation and inheritance. Over the past few years, a highly complex and diverse network of noncoding RNAs has been discovered. Research in animal models has shown that RNAs can be inherited and that RNA methyltransferases can be important for the transmission and expression of modified phenotypes in the next generation. We discuss possible mechanisms of RNA-mediated inheritance and the role of these mechanisms for human health and disease.