Francesca Tuorto

RNA methylation by Dnmt2 protects transfer RNAs against stress-induced cleavage

Dnmt2 proteins are the most conserved members of the DNA methyltransferase enzyme family, but their substrate specificity and biological functions have been a subject of controversy. We show here that, in addition to tRNA(Asp-GTC), tRNA(Val-AAC) and tRNA(Gly-GCC) are also methylated by Dnmt2. Drosophila Dnmt2 mutants showed reduced viability under stress conditions, and Dnmt2 relocalized to stress granules following heat shock. Strikingly, stress-induced cleavage of tRNAs was Dnmt2-dependent, and Dnmt2-mediated methylation protected tRNAs against ribonuclease cleavage. These results uncover a novel biological function of Dnmt2-mediated tRNA methylation, and suggest a role for Dnmt2 enzymes during the biogenesis of tRNA-derived small RNAs.

RNA cytosine methylation by Dnmt2 and NSun2 promotes tRNA stability and protein synthesis

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.

The mouse cytosine-5 RNA methyltransferase NSun2 is a component of the chromatoid body and required for testis differentiation.

ABSTRACT Posttranscriptional regulatory mechanisms are crucial for protein synthesis during spermatogenesis and are often organized by the chromatoid body. Here, we identify the RNA methyltransferase NSun2 as a novel component of the chromatoid body and, further, show that NSun2 is essential for germ cell differentiation in the mouse testis. In NSun2-depleted testes, genes encoding Ddx4, Miwi, and Tudor domain-containing (Tdr) proteins are repressed, indicating that RNA-processing and posttranscriptional pathways are impaired. Loss of NSun2 specifically blocked meiotic progression of germ cells into the pachytene stage, as spermatogonial and Sertoli cells were unaffected in knockout mice. We observed the same phenotype when we simultaneously deleted NSun2 and Dnmt2, the only other cytosine-5 RNA methyltransferase characterized to date, indicating that Dnmt2 was not functionally redundant with NSun2 in spermatogonial stem cells or Sertoli cells. Specific NSun2- and Dnmt2-methylated tRNAs decreased in abundance when both methyltransferases were deleted, suggesting that RNA methylation pathways play an essential role in male germ cell differentiation.

Hydroxylation of 5-methylcytosine by TET2 maintains the active state of the mammalian HOXA cluster

Differentiation is accompanied by extensive epigenomic reprogramming, leading to the repression of stemness factors and the transcriptional maintenance of activated lineage-specific genes. Here we use the mammalian Hoxa cluster of developmental genes as a model system to follow changes in DNA modification patterns during retinoic acid-induced differentiation. We find the inactive cluster to be marked by defined patterns of 5-methylcytosine (5mC). Upon the induction of differentiation, the active anterior part of the cluster becomes increasingly enriched in 5-hydroxymethylcytosine (5hmC), following closely the colinear activation pattern of the gene array, which is paralleled by the reduction of 5mC. Depletion of the 5hmC generating dioxygenase Tet2 impairs the maintenance of Hoxa activity and partially restores 5mC levels. Our results indicate that gene-specific 5mC-5hmC conversion by Tet2 is crucial for the maintenance of active chromatin states at lineage-specific loci.

The tRNA methyltransferase Dnmt2 is required for accurate polypeptide synthesis during haematopoiesis

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.

Extensive Methylation of Promoter Sequences Silences Lentiviral Transgene Expression During Stem Cell Differentiation In Vivo

Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.

Genome recoding by tRNA modifications

RNA modifications are emerging as an additional regulatory layer on top of the primary RNA sequence. These modifications are particularly enriched in tRNAs where they can regulate not only global protein translation, but also protein translation at the codon level. Modifications located in or in the vicinity of tRNA anticodons are highly conserved in eukaryotes and have been identified as potential regulators of mRNA decoding. Recent studies have provided novel insights into how these modifications orchestrate the speed and fidelity of translation to ensure proper protein homeostasis. This review highlights the prominent modifications in the tRNA anticodon loop: queuosine, inosine, 5-methoxycarbonylmethyl-2-thiouridine, wybutosine, threonyl–carbamoyl–adenosine and 5-methylcytosine. We discuss the functional relevance of these modifications in protein translation and their emerging role in eukaryotic genome recoding during cellular adaptation and disease.

Ribosomal transcription is regulated by PGC-1alpha and disturbed in Huntington’s disease

PGC-1α is a versatile inducer of mitochondrial biogenesis and responsive to the changing energy demands of the cell. As mitochondrial ATP production requires proteins that derive from translation products of cytosolic ribosomes, we asked whether PGC-1α directly takes part in ribosomal biogenesis. Here, we show that a fraction of cellular PGC-1α localizes to the nucleolus, the site of ribosomal transcription by RNA polymerase I. Upon activation PGC-1α associates with the ribosomal DNA and boosts recruitment of RNA polymerase I and UBF to the rDNA promoter. This induces RNA polymerase I transcription under different stress conditions in cell culture and mouse models as well as in healthy humans and is impaired already in early stages of human Huntington’s disease. This novel molecular link between ribosomal and mitochondrial biogenesis helps to explain sarcopenia and cachexia in diseases of neurodegenerative origin.

Queuosine‐modified tRNAs confer nutritional control of protein translation

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.

BisAMP: A web-based pipeline for targeted RNA cytosine-5 methylation analysis

RNA cytosine-5 methylation (m5C) has emerged as a key epitranscriptomic mark, which fulfills multiple roles in structural modulation, stress signaling and the regulation of protein translation. Bisulfite sequencing is currently the most accurate and reliable method to detect m5C marks at nucleotide resolution. Targeted bisulfite sequencing allows m5C detection at single base resolution, by combining the use of tailored primers with bisulfite treatment. A number of computational tools currently exist to analyse m5C marks in DNA bisulfite sequencing. However, these methods are not directly applicable to the analysis of RNA m5C marks, because DNA analysis focuses on CpG methylation, and because artifactual unconversion and misamplification in RNA can obscure actual methylation signals. We describe a pipeline designed specifically for RNA cytosine-5 methylation analysis in targeted bisulfite sequencing experiments. The pipeline is directly applicable to Illumina MiSeq (or equivalent) sequencing datasets using a web interface (https://bisamp.dkfz.de), and is defined by optimized mapping parameters and the application of tailored filters for the removal of artifacts. We provide examples for the application of this pipeline in the unambiguous detection of m5C marks in tRNAs from mouse embryonic stem cells and neuron-differentiated stem cells as well as in 28S rRNA from human fibroblasts. Finally, we also discuss the adaptability of BisAMP to the analysis of DNA methylation. Our pipeline provides an accurate, fast and user-friendly framework for the analysis of cytosine-5 methylation in amplicons from bisulfite-treated RNA.