Evaluation of the Properties of the DNA Methyltransferase from Aeropyrum pernix K1.

Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.

Reinforcement of transcriptional silencing by a positive feedback between DNA methylation and non-coding transcription.

Non-coding transcription is an important determinant of heterochromatin formation. In Arabidopsis thaliana a specialized RNA polymerase V (Pol V) transcribes pervasively and produces long non-coding RNAs. These transcripts work with small interfering RNA to facilitate locus-specific establishment of RNA-directed DNA methylation (RdDM). Subsequent maintenance of RdDM is associated with elevated levels of Pol V transcription. However, the impact of DNA methylation on Pol V transcription remained unresolved. We found that DNA methylation strongly enhances Pol V transcription. The level of Pol V transcription is reduced in mutants defective in RdDM components working downstream of Pol V, indicating that RdDM is maintained by a mutual reinforcement of DNA methylation and Pol V transcription. Pol V transcription is affected only on loci that lose DNA methylation in all sequence contexts in a particular mutant, including mutants lacking maintenance DNA methyltransferases, which suggests that RdDM works in a complex crosstalk with other silencing pathways.

Repression of CHROMOMETHYLASE 3 prevents epigenetic collateral damage in Arabidopsis.

DNA methylation has evolved to silence mutagenic transposable elements (TEs) while typically avoiding the targeting of endogenous genes. Mechanisms that prevent DNA methyltransferases from ectopically methylating genes are expected to be of prime importance during periods of dynamic cell cycle activities including plant embryogenesis. However, virtually nothing is known regarding how DNA methyltransferase activities are precisely regulated during embryogenesis to prevent the induction of potentially deleterious and mitotically stable genic epimutations. Here, we report that microRNA-mediated repression of CHROMOMETHYLASE 3 (CMT3) and the chromatin features that CMT3 prefers help prevent ectopic methylation of thousands of genes during embryogenesis that can persist for weeks afterwards. Our results are also consistent with CMT3-induced ectopic methylation of promoters or bodies of genes undergoing transcriptional activation reducing their expression. Therefore, the repression of CMT3 prevents epigenetic collateral damage on endogenous genes. We also provide a model that may help reconcile conflicting viewpoints regarding the functions of gene-body methylation that occurs in nearly all flowering plants.

Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli.

Targeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

CRISPR-based targeting of DNA methylation in Arabidopsis thaliana by a bacterial CG-specific DNA methyltransferase.

CRISPR-based targeted modification of epigenetic marks such as DNA cytosine methylation is an important strategy to regulate the expression of genes and their associated phenotypes. Although plants have DNA methylation in all sequence contexts (CG, CHG, CHH, where H = A, T, C), methylation in the symmetric CG context is particularly important for gene silencing and is very efficiently maintained through mitotic and meiotic cell divisions. Tools that can directly add CG methylation to specific loci are therefore highly desirable but are currently lacking in plants. Here we have developed two CRISPR-based CG-specific targeted DNA methylation systems for plants using a variant of the bacterial CG-specific DNA methyltransferase MQ1 with reduced activity but high specificity. We demonstrate that the methylation added by MQ1 is highly target specific and can be heritably maintained in the absence of the effector. These tools should be valuable both in crop engineering and in plant genetic research.

Ectopic targeting of CG DNA methylation in Arabidopsis with the bacterial SssI methyltransferase.

The ability to target epigenetic marks like DNA methylation to specific loci is important in both basic research and in crop plant engineering. However, heritability of targeted DNA methylation, how it impacts gene expression, and which epigenetic features are required for proper establishment are mostly unknown. Here, we show that targeting the CG-specific methyltransferase M.SssI with an artificial zinc finger protein can establish heritable CG methylation and silencing of a targeted locus in Arabidopsis. In addition, we observe highly heritable widespread ectopic CG methylation mainly over euchromatic regions. This hypermethylation shows little effect on transcription while it triggers a mild but significant reduction in the accumulation of H2A.Z and H3K27me3. Moreover, ectopic methylation occurs preferentially at less open chromatin that lacks positive histone marks. These results outline general principles of the heritability and interaction of CG methylation with other epigenomic features that should help guide future efforts to engineer epigenomes.

Genome-wide identification and analysis of DNA methyltransferase and demethylase gene families in Dendrobium officinale reveal their potential functions in polysaccharide accumulation.

BACKGROUND: DNA methylation is a conserved and important epigenetic modification involved in the regulation of numerous biological processes, including plant development, secondary metabolism, and response to stresses. However, no information is available regarding the identification of cytosine-5 DNA methyltransferase (C5-MTase) and DNA demethylase (dMTase) genes in the orchid Dendrobium officinale. RESULTS: In this study, we performed a genome-wide analysis of DoC5-MTase and DodMTase gene families in D. officinale. Integrated analysis of conserved motifs, gene structures and phylogenetic analysis showed that eight DoC5-MTases were divided into four subfamilies (DoCMT, DoDNMT, DoDRM, DoMET) while three DodMTases were divided into two subfamilies (DoDML3, DoROS1). Multiple cis-acting elements, especially stress-responsive and hormone-responsive ones, were found in the promoter region of DoC5-MTase and DodMTase genes. Furthermore, we investigated the expression profiles of DoC5-MTase and DodMTase in 10 different tissues, as well as their transcript abundance under abiotic stresses (cold and drought) and at the seedling stage, in protocorm-like bodies, shoots, and plantlets. Interestingly, most DoC5-MTases were downregulated whereas DodMTases were upregulated by cold stress. At the seedling stage, DoC5-MTase expression decreased as growth proceeded, but DodMTase expression increased. CONCLUSIONS: These results provide a basis for elucidating the role of DoC5-MTase and DodMTase in secondary metabolite production and responses to abiotic stresses in D. officinale.

The tumor suppressor Zinc finger protein 471 suppresses breast cancer growth and metastasis through inhibiting AKT and Wnt/ß-catenin signaling.

BACKGROUND: Zinc-finger protein 471 (ZNF471) is a member of the Krüppel-associated box domain zinc finger protein (KRAB-ZFP) family. ZNF471 is methylated in squamous cell carcinomas of tongue, stomach and esophageal. However, its role in breast carcinogenesis remains elusive. Here, we studied its expression, functions, and molecular mechanisms in breast cancer. METHODS: We examined ZNF471 expression by RT-PCR and qPCR. Methylation-specific PCR determined its promoter methylation. Its biological functions and related molecular mechanisms were assessed by CCK-8, clonogenicity, wound healing, Transwell, nude mice tumorigenicity, flow cytometry, BrdU-ELISA, immunohistochemistry and Western blot assays. RESULTS: ZNF471 was significantly downregulated in breast cell lines and tissues due to its promoter CpG methylation, compared with normal mammary epithelial cells and paired surgical-margin tissues. Ectopic expression of ZNF471 substantially inhibited breast tumor cell growth in vitro and in vivo, arrested cell cycle at S phase, and promoted cell apoptosis, as well as suppressed metastasis. Further knockdown of ZNF471 verified its tumor-suppressive effects. We also found that ZNF471 exerted its tumor-suppressive functions through suppressing epithelial-mesenchymal transition, tumor cell stemness and AKT and Wnt/ß-catenin signaling. CONCLUSIONS: ZNF471 functions as a tumor suppressor that was epigenetically inactivated in breast cancer. Its inhibition of AKT and Wnt/ß-catenin signaling pathways is one of the mechanisms underlying its anti-cancer effects.

Construction of a Universal and Label-Free Chemiluminescent Sensor for Accurate Quantification of Both Bacteria and Human Methyltransferases.

DNA methylation plays important roles in various biological processes, and the alteration of DNA methyltransferase activity can induce the aberrant DNA methylation patterns. Despite the progress in methyltransferase activity assays, few methods enable the detection of both bacteria and human methyltransferases. Herein, we construct a universal and label-free chemiluminescent sensor for accurate quantification of both bacteria methyltransferases (e.g., M. SssI methyltransferase (M.SssI MTase)) and human methyltransferases (e.g., DNA (cytosine-5)-methyltransferase 1, (Dnmt1)) by integrating a dumbbell probe with BssHII endonuclease-mediated rolling circle amplification (RCA). We ingeniously design a structure-switchable dumbbell probe which integrates target-recognition, BssHII endonuclease-cleavage, RCA amplification and signal transduction in one probe for the detection of both M.SssI MTase and Dnmt1. Moreover, the introduction of two BssHII endonuclease recognition sites in a dumbbell probe can greatly reduce the false positivity resulting from the incomplete cleavage of dumbbell probe by BssHII, because once one of two recognition sites is identified by BssHII, the dumbbell probe can be completely digested by Exonuclease III (Exo III) and Exonuclease I (Exo I) to prevent the nonspecific RCA. This chemiluminescent sensor can accurately quantify M.SssI MTase in both 10% serum and various cell lysis buffers, and even sensitively detect Dnmt1 activity in MCF-7 cells. Furthermore, this chemiluminescent sensor can be used to screen the inhibitors of Dnmt1 and M.SssI MTase, with promising applications in disease diagnosis and drug discovery.

DNA Geminivirus Infection Induces an Imprinted E3 Ligase Gene to Epigenetically Activate Viral Gene Transcription.

Flowering plants and mammals contain imprinted genes that are primarily expressed in the endosperm and placenta in a parent-of-origin manner. In this study, we show that early activation of the geminivirus genes C2 and C3 in Arabidopsis (Arabidopsis thaliana) plants, encoding a viral suppressor of RNA interference and a replication enhancer protein, respectively, is correlated with the transient vegetative expression of VARIANT IN METHYLATION5 (VIM5), an endosperm imprinted gene that is conserved in diverse plant species. VIM5 is a ubiquitin E3 ligase that directly targets the DNA methyltransferases MET1 and CMT3 for degradation by the ubiquitin-26S proteasome proteolytic pathway. Infection with Beet severe curly top virus induced VIM5 expression in rosette leaf tissues, possibly via the expression of the viral replication initiator protein, leading to the early activation of C2 and C3 coupled with reduced symmetric methylation in the C2-3 promoter and the onset of disease symptoms. These findings demonstrate how this small DNA virus recruits a host imprinted gene for the epigenetic activation of viral gene transcription. Our findings reveal a distinct strategy used by plant pathogens to exploit the host machinery in order to inhibit methylation-mediated defense responses when establishing infection.

KRAB-Induced Heterochromatin Effectively Silences PLOD2 Gene Expression in Somatic Cells and is Resilient to TGFß1 Activation.

Epigenetic editing, an emerging technique used for the modulation of gene expression in mammalian cells, is a promising strategy to correct disease-related gene expression. Although epigenetic reprogramming results in sustained transcriptional modulation in several in vivo models, further studies are needed to develop this approach into a straightforward technology for effective and specific interventions. Important goals of current research efforts are understanding the context-dependency of successful epigenetic editing and finding the most effective epigenetic effector(s) for specific tasks. Here we tested whether the fibrosis- and cancer-associated PLOD2 gene can be repressed by the DNA methyltransferase M.SssI, or by the non-catalytic Krüppel associated box (KRAB) repressor directed to the PLOD2 promoter via zinc finger- or CRISPR-dCas9-mediated targeting. M.SssI fusions induced de novo DNA methylation, changed histone modifications in a context-dependent manner, and led to 50%-70% reduction in PLOD2 expression in fibrotic fibroblasts and in MDA-MB-231 cancer cells. Targeting KRAB to PLOD2 resulted in the deposition of repressive histone modifications without DNA methylation and in almost complete PLOD2 silencing. Interestingly, both long-term TGFß1-induced, as well as unstimulated PLOD2 expression, was completely repressed by KRAB, while M.SssI only prevented the TGFß1-induced PLOD2 expression. Targeting transiently expressed dCas9-KRAB resulted in sustained PLOD2 repression in HEK293T and MCF-7 cells. Together, these findings point to KRAB outperforming DNA methylation as a small potent targeting epigenetic effector for silencing TGFß1-induced and uninduced PLOD2 expression.

Natural variation in DNA methylation homeostasis and the emergence of epialleles.

In plants and mammals, DNA methylation plays a critical role in transcriptional silencing by delineating heterochromatin from transcriptionally active euchromatin. A homeostatic balance between heterochromatin and euchromatin is essential to genomic stability. This is evident in many diseases and mutants for heterochromatin maintenance, which are characterized by global losses of DNA methylation coupled with localized ectopic gains of DNA methylation that alter transcription. Furthermore, we have shown that genome-wide methylation patterns in Arabidopsis thaliana are highly stable over generations, with the exception of rare epialleles. However, the extent to which natural variation in the robustness of targeting DNA methylation to heterochromatin exists, and the phenotypic consequences of such variation, remain to be fully explored. Here we describe the finding that heterochromatin and genic DNA methylation are highly variable among 725 A. thaliana accessions. We found that genic DNA methylation is inversely correlated with that in heterochromatin, suggesting that certain methylation pathway(s) may be redirected to genes upon the loss of heterochromatin. This redistribution likely involves a feedback loop involving the DNA methyltransferase, CHROMOMETHYLASE 3 (CMT3), H3K9me2, and histone turnover, as highly expressed, long genes with a high density of CMT3-preferred CWG sites are more likely to be methylated. Importantly, although the presence of CG methylation in genes alone may not affect transcription, genes containing CG methylation are more likely to become methylated at non-CG sites and silenced. These findings are consistent with the hypothesis that natural variation in DNA methylation homeostasis may underlie the evolution of epialleles that alter phenotypes.

Essential concepts are missing: Foreign DNA in food invades the organisms' cells and can lead to stochastic epigenetic alterations with a wide range of possible pathogenetic consequences.

In this article, a new concept for general pathogenesis has been proposed. Advances in molecular genetics have led to the realization that essential concepts in the framework of molecular biology are still missing. Clinical medicine is plagued by similar shortcomings: The questioning of current paradigms could open new vistas and invite challenging approaches. This article presents an unconventional idea. Foreign DNA which is regularly ingested with the essential food supply is not completely degraded. Small quantities of fragmented DNA rather persist transiently in the gastro-intestinal tract of mice and can be traced to various organ systems, except for cells in the germ line. Foreign DNA entering and persisting in mammalian cells can stochastically lead to genome-wide alterations of transcriptional and CpG DNA methylation profiles. In the course of food-ingested DNA invading somatic cells, completely new cell types can be generated which might be involved in the causation of common ailments. Projects emanating from this perception merit critical analysis and rigorous pursuit.

Microvesicles promote megakaryopoiesis by regulating DNA methyltransferase and methylation of Notch1 promoter.

Megakaryopoiesis is the process of formation of mature megakaryocytes that takes place in the bone marrow niche resulting in the release of platelets into the peripheral blood. It has been suggested that cell to cell communication in this dense bone marrow niche may influence the fate of the cells. Numerous studies point to the role of exosomes and microvesicles not only as a messenger of the cellular crosstalk but also in growth and developmental process of various cell types. In the current study, we explored the effects of megakaryocyte-derived microvesicles in hematopoietic cell lines in the context of differentiation. Our study demonstrated that microvesicles isolated from the induced megakaryocytic cell lines have the ability to stimulate noninduced cells specifically into that particular lineage. We showed that this lineage commencement comes from the change in the methylation status of Notch1 promoter, which is regulated by DNA methyltransferases.

Targeted DNA methylation of neurodegenerative disease genes via homology directed repair.

DNA methyltransferases (DNMTs) are thought to be involved in the cellular response to DNA damage, thus linking DNA repair mechanisms with DNA methylation. In this study we present Homology Assisted Repair Dependent Epigenetic eNgineering (HARDEN), a novel method of targeted DNA methylation that utilizes endogenous DNA double strand break repair pathways. This method allows for stable targeted DNA methylation through the process of homology directed repair (HDR) via an in vitro methylated exogenous repair template. We demonstrate that HARDEN can be applied to the neurodegenerative disease genes C9orf72 and APP, and methylation can be induced via HDR with both single and double stranded methylated repair templates. HARDEN allows for higher targeted DNA methylation levels than a dCas9-DNMT3a fusion protein construct at C9orf72, and genome-wide methylation analysis reveals no significant off-target methylation changes when inducing methylation via HARDEN, whereas the dCas9-DNMT3a fusion construct causes global off-target methylation. HARDEN is applied to generate a patient derived iPSC model of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD) that recapitulates DNA methylation patterns seen in patients, demonstrating that DNA methylation of the 5' regulatory region directly reduces C9orf72 expression and increases histone H3K9 tri-methylation levels.

Ectopic Methylation of a Single Persistently Unmethylated CpG in the Promoter of the Vitellogenin Gene Abolishes Its Inducibility by Estrogen through Attenuation of Upstream Stimulating Factor Binding.

The enhancer/promoter of the vitellogenin II gene (VTG) has been extensively studied as a model system of vertebrate transcriptional control. While deletion mutagenesis and in vivo footprinting identified the transcription factor (TF) binding sites governing its tissue specificity, DNase hypersensitivity and DNA methylation studies revealed the epigenetic changes accompanying its hormone-dependent activation. Moreover, upon induction with estrogen (E2), the region flanking the estrogen-responsive element (ERE) was reported to undergo active DNA demethylation. We now show that although the VTG ERE is methylated in embryonic chicken liver and in LMH/2A hepatocytes, its induction by E2 was not accompanied by extensive demethylation. In contrast, E2 failed to activate a VTG enhancer/promoter-controlled luciferase reporter gene methylated by SssI. Surprisingly, this inducibility difference could be traced not to the ERE but rather to a single CpG in an E-box (CACGTG) sequence upstream of the VTG TATA box, which is unmethylated in vivo but methylated by SssI. We demonstrate that this E-box binds the upstream stimulating factor USF1/2. Selective methylation of the CpG within this binding site with an E-box-specific DNA methyltransferase, Eco72IM, was sufficient to attenuate USF1/2 binding in vitro and abolish the hormone-induced transcription of the VTG gene in the reporter system.

A model of pulldown alignments from SssI-treated DNA improves DNA methylation prediction.

BACKGROUND: Protein pulldown using Methyl-CpG binding domain (MBD) proteins followed by high-throughput sequencing is a common method to determine DNA methylation. Algorithms have been developed to estimate absolute methylation level from read coverage generated by affinity enrichment-based techniques, but the most accurate one for MBD-seq data requires additional data from an SssI-treated Control experiment. RESULTS: Using our previous characterizations of Methyl-CpG/MBD2 binding in the context of an MBD pulldown experiment, we build a model of expected MBD pulldown reads as drawn from SssI-treated DNA. We use the program BayMeth to evaluate the effectiveness of this model by substituting calculated SssI Control data for the observed SssI Control data. By comparing methylation predictions against those from an RRBS data set, we find that BayMeth run with our modeled SssI Control data performs better than BayMeth run with observed SssI Control data, on both 100 bp and 10 bp windows. Adapting the model to an external data set solely by changing the average fragment length, our calculated data still informs the BayMeth program to a similar level as observed data in predicting methylation state on a pulldown data set with matching WGBS estimates. CONCLUSION: In both internal and external MBD pulldown data sets tested in this study, BayMeth used with our modeled pulldown coverage performs better than BayMeth run without the inclusion of any estimate of SssI Control pulldown, and is comparable to - and in some cases better than - using observed SssI Control data with the BayMeth program. Thus, our MBD pulldown alignment model can improve methylation predictions without the need to perform additional control experiments.

Post-transcriptional gene silencing triggers dispensable DNA methylation in gene body in Arabidopsis.

Spontaneous post-transcriptional silencing of sense transgenes (S-PTGS) is established in each generation and is accompanied by DNA methylation, but the pathway of PTGS-dependent DNA methylation is unknown and so is its role. Here we show that CHH and CHG methylation coincides spatially and temporally with RDR6-dependent products derived from the central and 3' regions of the coding sequence, and requires the components of the RNA-directed DNA methylation (RdDM) pathway NRPE1, DRD1 and DRM2, but not CLSY1, NRPD1, RDR2 or DCL3, suggesting that RDR6-dependent products, namely long dsRNAs and/or siRNAs, trigger PTGS-dependent DNA methylation. Nevertheless, none of these RdDM components are required to establish S-PTGS or produce a systemic silencing signal. Moreover, preventing de novo DNA methylation in non-silenced transgenic tissues grafted onto homologous silenced tissues does not inhibit the triggering of PTGS. Overall, these data indicate that gene body DNA methylation is a consequence, not a cause, of PTGS, and rule out the hypothesis that a PTGS-associated DNA methylation signal is transmitted independent of a PTGS signal.

Single-Molecule Fluorescence Imaging for Ultrasensitive DNA Methyltransferase Activity Measurement and Inhibitor Screening.

Aberrant DNA methylation by DNA methyltransferases (MTase) is related to the initiation and progression of many diseases. Thus, site-specific identification of DNA methylation and detection of MTase activity are very important to diagnose and treat methylation-related diseases. Herein, a single-molecule counting based ultrasensitive assay was developed for facile and direct detection of MTase activity and inhibitor screening without the assistance of restriction endonuclease. A double-strand DNA (dsDNA) was designed with the recognition site of M. SssI MTase and assembled on the coverslip surface. After the dsDNA was methylated by M. SssI, the biotin conjugated anti-5-methylcytosine antibody (5mC Ab) would specifically bind the CpG methylation site, and subsequently, the streptavidin-labeled quantum dots (QS585) bind the biotins. By taking and counting the image spots of fluorescently labeled methylated dsDNA molecules, the single-molecule imaging of methylated dsDNA molecules was recorded to quantify the DNA MTase activity. The spot number shows a linear relation with the logarithm of M. SssI concentration in the concentration range of 0.001-1 U/mL. Compared with most of the state of the art methods, the proposed assay displays a lower detection limit of 0.0005 U/mL and can detect the DNA MTase more directly. Moreover, it can selectively detect M. SssI in more complex samples. In addition, it is further demonstrated that the protocol could be successfully applied to evaluate the inhibition efficiency of M. SssI inhibitors. This assay is anticipated to provide a new approach for clinical diagnosis of methylation-related diseases and screening of new anticancer drugs.

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.