Eukaryotic 5-methylcytosine (m5C) RNA Methyltransferases: Mechanisms, Cellular Functions, and Links to Disease

Genes, Journal Year: 2019, Volume and Issue: 10(2), P. 102 - 102

Published: Jan. 30, 2019

5-methylcytosine (m⁵C) is an abundant RNA modification that's presence reported in a wide variety of species, including cytoplasmic and mitochondrial ribosomal RNAs (rRNAs) transfer (tRNAs), as well messenger (mRNAs), enhancer (eRNAs) number non-coding RNAs. In eukaryotes, C5 methylation cytosines catalyzed by enzymes the NOL1/NOP2/SUN domain (NSUN) family, DNA methyltransferase homologue DNMT2. recent years, substrate target nucleotides for each these methyltransferases have been identified, structural biochemical analyses provided first insights into how achieves specificity. Functional characterizations proteins modifications they install revealed important roles diverse aspects both nuclear gene expression. Importantly, this knowledge has enabled better understanding molecular basis diseases caused mutations genes encoding m⁵C or changes expression level enzymes.

Language: Английский

---

Widespread adenine N6-methylation of active genes in fungi

Nature Genetics, Journal Year: 2017, Volume and Issue: 49(6), P. 964 - 968

Published: May 8, 2017

Language: Английский

---

The role of DNA methylation in epigenetics of aging

Pharmacology & Therapeutics, Journal Year: 2018, Volume and Issue: 195, P. 172 - 185

Published: Nov. 9, 2018

Recent research suggests that epigenetics, especially DNA methylation, plays a mechanistic role in aging. Epigenetic clocks, which measure changes few hundred specific CpG sites, can accurately predict chronological age variety of species, including humans. These clocks are currently the best biomarkers for predicting mortality Additionally, several studies have characterized effects aging across methylome wide tissues from humans and mice. A small fraction (~2%) sites show age-related changes, either hypermethylation or hypomethylation with Evaluation non-CpG site methylation has only been examined studies, about ~0.5% these showing change age. Therefore, while cytosines genome age, this represents 2 to 3 million genome. Importantly, study compare effect on male female mice found >95% hippocampus were sexually divergent, i.e., did not differ between males females at young but occurred one sex other. The tend be enriched under-represented genomic contexts, some commonalities species require further investigation. strongest evidence play comes anti-aging interventions (e.g., caloric restriction, dwarfism, rapamycin treatment) deaccelerate epigenetic reverse/prevent 20 40% methylation. It will important future demonstrate least directly lead alterations transcriptome cells/tissues could potentially contribute

Language: Английский

---

NSUN 3 and ABH 1 modify the wobble position of mt‐t RNA ^Met to expand codon recognition in mitochondrial translation

The EMBO Journal, Journal Year: 2016, Volume and Issue: 35(19), P. 2104 - 2119

Published: Aug. 6, 2016

Article6 August 2016Open Access NSUN3 and ABH1 modify the wobble position of mt-tRNAMet to expand codon recognition in mitochondrial translation Sara Haag Institute for Molecular Biology, University Medical Center Göttingen, Georg-August-University, Germany Search more papers by this author Katherine E Sloan Namit Ranjan Department Physical Biochemistry, Max Planck Biophysical Chemistry, Ahmed S Warda Jens Kretschmer Charlotte Blessing Benedikt Hübner Jan Seikowski Organic Biomolecular Sven Dennerlein Cellular Peter Rehling Göttingen Centre Biosciences, Marina V Rodnina Claudia Höbartner Corresponding Author [email protected] orcid.org/0000-0001-7063-5456 Markus T Bohnsack Information Haag1,‡, Sloan1,‡, Ranjan2,‡, Warda1,‡, Kretschmer1, Blessing1, Hübner1, Seikowski3,4, Dennerlein5, Rehling4,5,6, Rodnina2, *,3 *,1,6 1Institute 2Department 3Institute 4Max 5Institute 6Göttingen ‡These authors contributed equally work *Corresponding author. Tel: +49 551 395968; Fax: 395960; E-mail: 3920906; 3921712; The EMBO Journal (2016)35:2104-2119https://doi.org/10.15252/embj.201694885 See also: F Boos et al (October 2016) [The copyright line article was changed on 1 October 2016 after original online publication.] PDFDownload PDF text main figures. Peer ReviewDownload a summary editorial decision process including letters, reviewer comments responses feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitochondrial gene expression uses non-universal genetic code mammals. Besides reading conventional AUG codon, (mt-)tRNAMet mediates incorporation methionine AUA AUU codons during initiation elongation. We show that RNA methyltransferase localises mitochondria interacts with methylate cytosine 34 (C34) at position. specifically recognises anticodon stem loop (ASL) tRNA, explaining why mutation compromises ASL basepairing leads disease. further identify ALKBH1/ABH1 as dioxygenase responsible oxidising m5C34 generate an f5C34 modification. In vitro studies factors reveal preferential utilisation initiation. Depletion either or strongly affects human cells, implying modifications generated both enzymes are necessary function. Together, our data how sequential action ABH1, allowing single tRNAMet recognise different encoding methionine. Synopsis acts tRNAMet, be recognised tRNA offering insight consequence reported disease mutations. introduces 5-methylcytosine modification tRNAMet. can oxidised ABH1/ALKBH1 mt-tRNAMet. These "wobble position" installed required efficient translation. pathway enables three used mitochondria. requires stable stem-loop methylation, mutations disrupt lead Introduction More than hundred chemical ribonucleosides have been identified cellular RNAs (Czerwoniec al, 2009; Motorin Helm, 2011). Modifications regulate biogenesis, structure function corresponding RNA–protein complexes (RNPs). Many occur involved therefore likely affect protein synthesis. Several modified 6-methyladenosine (m6A), 5-methylcytidine (m5C), 1-methyladenosine (m1A) pseudouridine recently shown messenger (m)RNAs their stability (see e.g. Carlile 2014; Liu Jia, Dominissini 2016). Methylated nucleosides undergo proteins AlkB family alpha-ketoglutarate Fe(II)-dependent dioxygenases (ALKBH1-8 FTO cells) oxidise even remove DNA (Fedeles 2015; Ougland 2015), increasing dynamics regulation roles metabolism. Compared mRNAs other RNAs, transfer (t)RNAs ribosomal (r)RNAs contain highest proportion nucleosides. large majority rRNA already co-transcriptionally small nucleolar (sno)RNPs, only few base require lone-standing (Watkins Bohnsack, 2012; Sharma Lafontaine, 2015). tRNAs largest variety nucleoside modifications, many them suggested biogenesis nuclear export, structure, interaction aminoacyl-tRNA-sythetases (Agris 2007; Leisegang Hori, Duechler 2016; Rodnina, (the position"). modulate codon–anticodon basepairing, often one several third codon. Mutations introducing these base" alterations sequences such associated disease, especially (Lott 2013; Powell One ribonucleoside has tRNAs, cytoplasmic rRNA, non-coding is (m5C). m5C any seven Nol1/Nop2/SUN domain (NSUN) enzyme named 2 (DNMT2). DNMT2 mainly catalyses 38 tRNAAsp cells (Goll 2006), while so far characterised NSUN specificity (NSUN2, NSUN6; Schaefer 2010; Tuorto Blanco 2015a) (NSUN1/NOP2, NSUN5; Tafforeau Schosserer NSUN2 also vault (Hussain 2013), NSUN4 described localise where it 12S mice (Cámara 2011; Metodiev 2014). last uncharacterised members family, we here matrix cells. Using vivo UV cross-linking analysis cDNA (CRAC) 5-azacytidine (5-AzaC) CRAC, (m5C) position". addition, find ALKBH1/ABH1, generating 5-formylcytidine (f5C) Analysis synthesised revealed system utilising elongation depends state C34 vivo, knock-down abolishes formation, depletion decrease Furthermore, reducing levels significant suggesting important two Interestingly, substrate pathogenic lack Results 10 years ago computational member Nol1/Nop2/Sun putative methyltransferases (Bujnicki 2004). (Rhee 2013); however, target spectrum biological remained unknown. To confirm localisation NSUN3, HEK293 cell stably expressing NSUN3-GFP from tetracycline-inducible promoter. Confocal fluorescence microscopy distinct foci showed co-localisation mitotracker (Fig 1A), indicating NSUN3. determine whether imported into mitochondria, performed protease protection assays using NSUN3-HisPrcFLAG (Hexahistidine-PreScission cleavage site-2×FLAG tagged NSUN3) line. isolated were then left intact, subjected swelling rupture outer membrane mitoplasts disrupted sonication before treatment concentrations proteinase K. While intact led degradation TOM70, intermembrane space TIM23 digested mitoplasts. Similar matrix-localised TIM44, became susceptible K digestion upon 1B), localised Figure 1. analysed NSUN3-GFP. (green) staining Mitotracker (red) separately overlay DAPI indicate nuclei. scale bar represents 5 μm. analyse submitochondrial untreated, swollen hypotonic buffer (Mitoplasts) (Sonic.) amounts (PK) indicated, followed SDS–PAGE Western blotting antibodies against TIM23, TOM70 FLAG-tagged Note TIM44 extends matrix, N-terminus largely exposed surface. asterisk indicates cross-reaction antibody. Download figure PowerPoint associates (CRAC; 2015) experiments line; HisPrcFLAG tag control. treated cytidine derivative reagent, which incorporated nascent traps nucleotides covalent protein–RNA intermediate methylation reaction 2A; Khoddami Cairns, 2013). Without 5-AzaC purified trimming, radiolabelling ligation adaptors bound RNA. Protein–RNA separated SDS–PAGE, transferred X-ray film. Both resulted strong specific signal not observed controls 2B), association RNAs. Interacting extracted RT–PCR library Illumina deep sequencing. Mapping obtained sequence reads genome over-representation mitochondrial-encoded (mt-RNA). mt-RNA represented 40% 62% total respectively, compared 4% control (Figs 2C–E EV1A), As enriched NSUN3-cross-linked fractions 2D E, lower panels) 2C, panel), distribution between 22 tRNAs. Strikingly, 50 95% mapped experiments, respectively 2F). contrast, contained 5% sequencing mt-tRNAMet, interaction, control, catalytically inactive NSUN3(C265A)-HisPrcFLAG mutant, catalytic cysteine TCT tripeptide conserved motif IV replaced alanine (C265A). After isolation via proteins, interacting Northern probes detection mt-tRNAPro, mt-tRNAGlu 2G). mt-tRNAPro could detected eluates, eluate wild-type sample, but controls, supporting requirement containing suggest active mechanism mediate its 2. cross-links A. Structure formation adduct. B. (NSUN3) alone (FLAG) cross-linked (−), (UV) (5-AzaC). affinity trimmed, end-labelled 32P phosphate ligated linkers. nitrocellulose C–E. (D, E) FLAG (C) samples (B). membrane-bound converted production Pie charts present classes relative mapping genome. Bar graphs below (mt-)tRNA, mt-rRNA mt-mRNA among Abbreviations: RNA; snRNA, snoRNA, mtRNA, miscRNA, miscellaneous miRNA, microRNA; lncRNA, long F. Relative CRAC (FLAG). Only mt-tRNAs all labelled. G. mutant (C265A) blot mt-tRNAGlu. Inputs (0.1%) eluates (50%) right. Click figure. EV1. Met-i Met-e do represent substrates A, Fig (A) percentages individual given graphically each sample. (B) shown. C. reactions recombinant His14-MBP-NSUN3 His14-MBP-NSUN3-C265A (C265A), [3H-methyl]-labelled S-adenosylmethionine methyl group donor vitro-transcribed tRNAiMet tRNAeMet. denaturing polyacrylamide gel, stained ethidium bromide (EtBr) inputs film (3H-Me). D. RNA-associated (A). mt-tRNAiMet mt-tRNAeMet. panel identical 2G. E. nucleotide loops (left) (right) methylates gain activity prepared (NSUN3-C265A) T7 RNA-polymerase transcripts presence S-[3H-methyl] adenosylmethionine (SAM) donor. efficiently methylated transcripts, abolished 3A). 3. modifies 3H-labelled along (light grey) (dark per million reads. indicated bar. Cloverleaf scheme sequence. Nucleosides exchanged mutational following panels marked arrows, positions given. mutants region (C). Two exposure times films 16 h (short) 3 days (long). assay chemically experiment enrichment sets, had (tRNAiMet) (tRNAeMet) over-represented (8% control; 18% 79% cross-linking; EV1B). tested tRNAeMet assays. very weak no EV1C). possible interactions immunoprecipitation alone, co-precipitation blotting. EV1D), does bind occurred lysis due similar EV1E). Together 1), weakly vitro, rather tRNAiMet, genuine vivo. order read density 3B) residue lies within region. individually mutated adenosine (ASL cytosines) uracil (cytosines ASL; 3C). Although cytosines affected NSUN3-mediated assays, 3D), This conclusion confirmed when 3E), finding generates moiety Among C39U previously patients dysfunction Tang particularly poor might critical recognition. distinguish possibilities, series u

Language: Английский

---

Epigenetics of Modified DNA Bases: 5-Methylcytosine and Beyond

Frontiers in Genetics, Journal Year: 2018, Volume and Issue: 9

Published: Dec. 18, 2018

Modification of DNA bases plays vital roles in the epigenetic control gene expression both animals and plants. Though much attention is given to conventional signature 5-methylcytosine (5-mC), field epigenetics attracting increased scientific interest through discovery additional modifications their controlling expression. Theoretically, each can be modified; however, cytosine adenine only are known so far. This review focuses on recent findings well-studied yet poorly characterized modification which serve as an layer regulation discuss potential Cytosine at symmetric (CG, CHG) asymmetric (CHH) contexts a key feature. In addition ROS1 family mediated demethylation, Ten-Eleven Translocation proteins-mediated hydroxylation 5-mC 5-hydroxymethylcytosine active demethylation pathway also discussed. The marks associated with several cellular developmental processes, pluripotency stem cells, neuron cell development, tumor development animals. Therefore, most recently discovered N6-methyladenine, mark regulatory potential, described. Interestingly, these newly found genomes lack canonical 5-mC, signifying independent functions. These modified considered important players epigenomics. for combinatorial interaction among suggests that codon likely substantially more complicated than it thought today.

Language: Английский

---

Epigenetics and Trained Immunity

Antioxidants and Redox Signaling, Journal Year: 2017, Volume and Issue: 29(11), P. 1023 - 1040

Published: Oct. 5, 2017

A growing body of clinical and experimental evidence has challenged the traditional understanding that only adaptive immune system can mount immunological memory. Recent findings describe characteristics innate system, underscored by its ability to remember antecedent foreign encounters respond in a nonspecific sensitized manner reinfection. This been termed trained immunity. Although beneficial context recurrent infections, this might actually contribute chronic immune-mediated diseases, such as atherosclerosis. Advances: In line with proposed role sustaining cellular memories, epigenetic reprogramming emerged critical determinant technological computational advances improve unbiased acquisition epigenomic profiles have significantly enhanced our appreciation for complexities chromatin architecture contexts diverse challenges.Key resolving distinct signatures memory is comprehensive precise physiological targets regulatory proteins recognize, deposit, remove chemical modifications from well other gene-regulating factors. Drawing rapidly expanding compendium studies, review details current perspective pathways support adapted phenotypes monocytes macrophages.We explore future strategies are aimed at exploiting mechanism immunity prevention treatment infections disorders.

Language: Английский

---

Direct RNA nanopore sequencing of full-length coronavirus genomes provides novel insights into structural variants and enables modification analysis

Genome Research, Journal Year: 2019, Volume and Issue: 29(9), P. 1545 - 1554

Published: Aug. 22, 2019

Sequence analyses of RNA virus genomes remain challenging owing to the exceptional genetic plasticity these viruses. Because high mutation and recombination rates, genome replication by viral RNA-dependent polymerases leads populations closely related viruses, so-called "quasispecies." Standard (short-read) sequencing technologies are ill-suited reconstruct large numbers full-length haplotypes (1) (2) subgenome-length (sg) RNAs composed noncontiguous regions. Here, we used a full-length, direct (DRS) approach based on nanopores characterize produced in cells infected with human coronavirus. By using DRS, were able map longest (∼26-kb) contiguous read reference genome. combining Illumina Oxford Nanopore sequencing, reconstructed highly accurate consensus sequence coronavirus (HCoV)-229E (27.3 kb). Furthermore, long reads that did not require an assembly step, identify, cells, diverse novel HCoV-229E sg be characterized. Also, DRS approach, which circumvents reverse transcription amplification RNA, allowed us detect methylation sites RNAs. Our work paves way for haplotype-based quasispecies showing feasibility intra-sample haplotype separation. Even though several technical challenges addressed exploit potential nanopore technology fully, our illustrates may significantly advance genomic studies complex populations, including predictions long-range interactions individual haplotypes.

Language: Английский

---

The Roles of Human DNA Methyltransferases and Their Isoforms in Shaping the Epigenome

Genes, Journal Year: 2019, Volume and Issue: 10(2), P. 172 - 172

Published: Feb. 23, 2019

A DNA sequence is the hard copy of human genome and it a driving force in determining physiological processes an organism. Concurrently, chemical modification its related histone proteins dynamically involved regulating diseases, which overall constitutes epigenome network. Among various forms epigenetic modifications, methylation at C-5 position cytosine cytosine⁻guanine (CpG) dinucleotide one most well studied modifications. methyltransferases (DNMTs) are family enzymes generating maintaining CpG across genome. In mammalian systems, performed by DNMT1 DNMT3s (DNMT3A 3B). predominantly maintenance during cell division, while establishing de novo both embryonic somatic cells. general, all DNMTs require accessory proteins, such as ubiquitin-like containing plant homeodomain (PHD) really interesting new gene (RING) finger domain 1 (UHRF1) or DNMT3-like (DNMT3L), for their biological function. This review mainly focuses on role DNMT3B isoforms methylation, especially with respect to protein.

Language: Английский

---

The role of RNA m⁵C modification in cancer metastasis

International Journal of Biological Sciences, Journal Year: 2021, Volume and Issue: 17(13), P. 3369 - 3380

Published: Jan. 1, 2021

Epigenetic modification plays a crucial regulatory role in the biological processes of eukaryotic cells.The recent characterization DNA and RNA methylation is still ongoing.Tumor metastasis has long been an unconquerable feature fight against cancer.As inevitable component epigenetic network, 5-methylcytosine associated with multifarious cellular systemic diseases, including cell migration cancer metastasis.Recently, gratifying progress achieved determining molecular interactions between m 5 C writers (DNMTs NSUNs), demethylases (TETs), readers (YTHDF2, ALYREF YBX1) RNAs.However, underlying mechanism mobility remains unclear.The functions are believed to regulate gene expression at post-transcription level involved metabolism movement.In this review, we emphatically summarize updates on components related networks.The content will be focused potential mechanisms diseases.We discuss relevant upstream downstream interacting molecules their associations metastasis.

Language: Английский

---

DNA 5-methylcytosine detection and methylation phasing using PacBio circular consensus sequencing

Nature Communications, Journal Year: 2023, Volume and Issue: 14(1)

Published: July 8, 2023

Long single-molecular sequencing technologies, such as PacBio circular consensus (CCS) and nanopore sequencing, are advantageous in detecting DNA 5-methylcytosine CpGs (5mCpGs), especially repetitive genomic regions. However, existing methods for 5mCpGs using CCS less accurate robust. Here, we present ccsmeth, a deep-learning method to detect reads. We sequence polymerase-chain-reaction treated M.SssI-methyltransferase of one human sample training ccsmeth. Using long (≥10 Kb) reads, ccsmeth achieves 0.90 accuracy 0.97 Area Under the Curve on 5mCpG detection at single-molecule resolution. At genome-wide site level, >0.90 correlations with bisulfite only 10× Furthermore, develop Nextflow pipeline, ccsmethphase, haplotype-aware methylation then Chinese family trio validate it. ccsmethphase can be robust tools 5-methylcytosines.

Language: Английский

---