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In Figure 5, translation initiation is promoted not by the indicated protein, but by YTHDF1 (see below).
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N(6)-methyladenosine (m(6)A) is the most abundant internal modification in mammalian mRNA. This modification is reversible and non-stoichiometric and adds another layer to the dynamic control of mRNA metabolism. The stability of m(6)A-modified mRNA is regulated by an m(6)A reader protein, human YTHDF2, which recognizes m(6)A and reduces the stability of target transcripts. Looking at additional functional roles for the modification, we find that another m(6)A reader protein, human YTHDF1, actively promotes protein synthesis by interacting with translation machinery. In a unified mechanism of m(6)A-based regulation in the cytoplasm, YTHDF2-mediated degradation controls the lifetime of target transcripts, whereas YTHDF1-mediated translation promotion increases translation efficiency, ensuring effective protein production from dynamic transcripts that are marked by m(6)A. Therefore, the m(6)A modification in mRNA endows gene expression with fast responses and controllable protein production through these mechanisms.
Assuntos
Adenosina/análogos & derivados , Adenosina/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas , Humanos , Fatores de Iniciação de Peptídeos/metabolismo , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismoRESUMO
The recent discovery of reversible mRNA methylation has opened a new realm of post-transcriptional gene regulation in eukaryotes. The identification and functional characterization of proteins that specifically recognize RNA N6-methyladenosine (m6A) unveiled it as a modification that cells utilize to accelerate mRNA metabolism and translation. N6-adenosine methylation directs mRNAs to distinct fates by grouping them for differential processing, translation and decay in processes such as cell differentiation, embryonic development and stress responses. Other mRNA modifications, including N1-methyladenosine (m1A), 5-methylcytosine (m5C) and pseudouridine, together with m6A form the epitranscriptome and collectively code a new layer of information that controls protein synthesis.
Assuntos
Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , 5-Metilcitosina/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Ciclo Celular/genética , Diferenciação Celular/genética , Ritmo Circadiano/genética , Regulação da Expressão Gênica , Humanos , Metilação , Conformação de Ácido Nucleico , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
DNA and histone modifications have notable effects on gene expression1. Being the most prevalent internal modification in mRNA, the N6-methyladenosine (m6A) mRNA modification is as an important post-transcriptional mechanism of gene regulation2-4 and has crucial roles in various normal and pathological processes5-12. However, it is unclear how m6A is specifically and dynamically deposited in the transcriptome. Here we report that histone H3 trimethylation at Lys36 (H3K36me3), a marker for transcription elongation, guides m6A deposition globally. We show that m6A modifications are enriched in the vicinity of H3K36me3 peaks, and are reduced globally when cellular H3K36me3 is depleted. Mechanistically, H3K36me3 is recognized and bound directly by METTL14, a crucial component of the m6A methyltransferase complex (MTC), which in turn facilitates the binding of the m6A MTC to adjacent RNA polymerase II, thereby delivering the m6A MTC to actively transcribed nascent RNAs to deposit m6A co-transcriptionally. In mouse embryonic stem cells, phenocopying METTL14 knockdown, H3K36me3 depletion also markedly reduces m6A abundance transcriptome-wide and in pluripotency transcripts, resulting in increased cell stemness. Collectively, our studies reveal the important roles of H3K36me3 and METTL14 in determining specific and dynamic deposition of m6A in mRNA, and uncover another layer of gene expression regulation that involves crosstalk between histone modification and RNA methylation.
Assuntos
Adenosina/análogos & derivados , Histonas/química , Histonas/metabolismo , Lisina/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transcrição Gênica , Adenosina/metabolismo , Animais , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Humanos , Lisina/química , Metilação , Metiltransferases/deficiência , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , RNA Polimerase II/metabolismo , Elongação da Transcrição Genética , Transcriptoma/genéticaRESUMO
In this issue of Molecular Cell, Ivanova et al. (2017) report key functions of the m6A reader YTHDF2 in the regulation of mammalian development during oocyte maturation and early zygotic development.
Assuntos
Células Germinativas , Zigoto , Animais , Feminino , Fertilidade , Humanos , Oócitos , OogêneseRESUMO
The maternal-to-zygotic transition (MZT) is one of the most profound and tightly orchestrated processes during the early life of embryos, yet factors that shape the temporal pattern of vertebrate MZT are largely unknown. Here we show that over one-third of zebrafish maternal messenger RNAs (mRNAs) can be N6-methyladenosine (m6A) modified, and the clearance of these maternal mRNAs is facilitated by an m6A-binding protein, Ythdf2. Removal of Ythdf2 in zebrafish embryos decelerates the decay of m6A-modified maternal mRNAs and impedes zygotic genome activation. These embryos fail to initiate timely MZT, undergo cell-cycle pause, and remain developmentally delayed throughout larval life. Our study reveals m6A-dependent RNA decay as a previously unidentified maternally driven mechanism that regulates maternal mRNA clearance during zebrafish MZT, highlighting the critical role of m6A mRNA methylation in transcriptome switching and animal development.
Assuntos
Adenosina/análogos & derivados , Desenvolvimento Embrionário/genética , Estabilidade de RNA , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Zigoto/metabolismo , Adenosina/metabolismo , Animais , Feminino , Masculino , RNA Mensageiro Estocado/química , RNA Mensageiro Estocado/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Tempo , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismoRESUMO
RNA-protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA-protein interactions in living cells in an unbiased manner. We used two approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N6-methyladenosine (m6A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m6A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA-protein interactions in living cells.
Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Enzimas Multifuncionais/metabolismo , Mapeamento de Interação de Proteínas/métodos , RNA/metabolismo , Telomerase/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Biotinilação , Sistemas CRISPR-Cas , Metilação de DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Células HEK293 , Humanos , Espectrometria de Massas , Enzimas Multifuncionais/genética , Ligação Proteica , RNA/genética , Telomerase/genéticaRESUMO
In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos/genética , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Citosina/análogos & derivados , Citosina/metabolismo , Proteínas de Ligação a DNA/genética , Dioxigenases , Camundongos , Camundongos Knockout , Oxirredução , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas/genética , Análise de Sequência de DNA , Transcriptoma/genética , Dedos de Zinco/genéticaRESUMO
Genetic variants associated with autism spectrum disorders (ASDs) are enriched in genes encoding synaptic proteins and chromatin regulators. Although the role of synaptic proteins in ASDs is widely studied, the mechanism by which chromatin regulators contribute to ASD risk remains poorly understood. Upon profiling and analyzing the transcriptional and epigenomic features of genes expressed in the cortex, we uncovered a unique set of long genes that contain broad enhancer-like chromatin domains (BELDs) spanning across their entire gene bodies. Analyses of these BELD genes show that they are highly transcribed with frequent RNA polymerase II (Pol II) initiation and low Pol II pausing, and they exhibit frequent chromatin-chromatin interactions within their gene bodies. These BELD features are conserved from rodents to humans, are enriched in genes involved in synaptic function, and appear post-natally concomitant with synapse development. Importantly, we find that BELD genes are highly implicated in neurodevelopmental disorders, particularly ASDs, and that their expression is preferentially down-regulated in individuals with idiopathic autism. Finally, we find that the transcription of BELD genes is particularly sensitive to alternations in ASD-associated chromatin regulators. These findings suggest that the epigenomic regulation of BELD genes is important for post-natal cortical development and lend support to a model by which mutations in chromatin regulators causally contribute to ASDs by preferentially impairing BELD gene transcription.
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Transtorno do Espectro Autista/genética , Cromatina/genética , Sequências Reguladoras de Ácido Nucleico/genética , Animais , Transtorno Autístico/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Neurogênese/genética , RNA Polimerase II/genética , Transcrição Gênica/genéticaRESUMO
DNA 5-methylcytosine (5mC)-specific mapping has been hampered by severe DNA degradation and the presence of 5-hydroxymethylcytosine (5hmC) using the conventional bisulfite sequencing approach. Here, we present a 5mC-specific whole-genome amplification method (5mC-WGA), with which we achieved 5mC retention during DNA amplification from limited input down to 10 pg scale with limited interference from 5hmC signals, providing DNA 5mC methylome with high reproducibility and accuracy.
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5-Metilcitosina/química , DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Análise de Sequência de DNA/métodos , Animais , DNA/química , Metilação de DNA , Humanos , Camundongos , Sulfitos/química , Sequenciamento Completo do GenomaRESUMO
The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper(II) as a natural signal to cope with stress caused by antibiotics or the environment.
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Cobre/metabolismo , Resistência Microbiana a Medicamentos , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Repressoras/metabolismo , Modelos Moleculares , Transdução de SinaisAssuntos
Precursores de RNA , RNA , Adenosina/análogos & derivados , Adenosina/química , Éxons , Metilação , RNA Mensageiro/químicaRESUMO
Prof. Chuan He was awarded the Tetrahedron Prize this year, one of the world's most prestigious prizes in organic chemistry. This In Focus briefly delves into the remarkable work of Prof. Chuan He and explores how his recent accolades underscore his impact on the world of science. His seminal contributions have paved the way for new directions at the interface of organic chemistry and life sciences.
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Distinções e Prêmios , QuímicaRESUMO
The transcriptional regulatory protein OhrR is converted into a fluorescent bioprobe capable of detecting organic hydroperoxides in living cells with high sensitivity and selectivity.
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Proteínas de Bactérias/metabolismo , Corantes Fluorescentes/metabolismo , Peróxido de Hidrogênio/metabolismo , Imagem Molecular/métodos , Compostos Orgânicos/metabolismo , Engenharia de Proteínas , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sobrevivência Celular , Corantes Fluorescentes/química , Células HeLa , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Especificidade por SubstratoRESUMO
Internal N6-methyladenosine (m6A) modification is one of the most common and abundant modifications of RNA. However, the biological roles of viral RNA m6A remain elusive. Here, using human metapneumovirus (HMPV) as a model, we demonstrate that m6A serves as a molecular marker for innate immune discrimination of self from non-self RNAs. We show that HMPV RNAs are m6A methylated and that viral m6A methylation promotes HMPV replication and gene expression. Inactivating m6A addition sites with synonymous mutations or demethylase resulted in m6A-deficient recombinant HMPVs and virion RNAs that induced increased expression of type I interferon, which was dependent on the cytoplasmic RNA sensor RIG-I, and not on melanoma differentiation-associated protein 5 (MDA5). Mechanistically, m6A-deficient virion RNA induces higher expression of RIG-I, binds more efficiently to RIG-I and facilitates the conformational change of RIG-I, leading to enhanced interferon expression. Furthermore, m6A-deficient recombinant HMPVs triggered increased interferon in vivo and were attenuated in cotton rats but retained high immunogenicity. Collectively, our results highlight that (1) viruses acquire m6A in their RNA as a means of mimicking cellular RNA to avoid detection by innate immunity and (2) viral RNA m6A can serve as a target to attenuate HMPV for vaccine purposes.
Assuntos
Adenosina/análogos & derivados , Proteína DEAD-box 58/genética , Evasão da Resposta Imune/genética , Interferon beta/genética , Metapneumovirus/imunologia , RNA Viral/genética , Células A549 , Adenosina/imunologia , Adenosina/metabolismo , Animais , Chlorocebus aethiops , Proteína DEAD-box 58/imunologia , Regulação da Expressão Gênica , Genoma Viral/imunologia , Células HeLa , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Fator Regulador 3 de Interferon/genética , Fator Regulador 3 de Interferon/imunologia , Helicase IFIH1 Induzida por Interferon/genética , Helicase IFIH1 Induzida por Interferon/imunologia , Interferon beta/imunologia , Metapneumovirus/genética , Metapneumovirus/crescimento & desenvolvimento , NF-kappa B/genética , NF-kappa B/imunologia , Infecções por Paramyxoviridae/genética , Infecções por Paramyxoviridae/imunologia , Infecções por Paramyxoviridae/virologia , RNA Viral/imunologia , Receptores Imunológicos , Sigmodontinae , Transdução de Sinais , Células THP-1 , Células Vero , Vírion/genética , Vírion/crescimento & desenvolvimento , Vírion/imunologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
N6-methyladenosine (m6A) is the most prevalent internal modification of mRNAs in most eukaryotes. Here we show that RNAs of human respiratory syncytial virus (RSV) are modified by m6A within discreet regions and that these modifications enhance viral replication and pathogenesis. Knockdown of m6A methyltransferases decreases RSV replication and gene expression whereas knockdown of m6A demethylases has the opposite effect. The G gene transcript contains the most m6A modifications. Recombinant RSV variants expressing G transcripts that lack particular clusters of m6A display reduced replication in A549 cells, primary well differentiated human airway epithelial cultures, and respiratory tracts of cotton rats. One of the m6A-deficient variants is highly attenuated yet retains high immunogenicity in cotton rats. Collectively, our results demonstrate that viral m6A methylation upregulates RSV replication and pathogenesis and identify viral m6A methylation as a target for rational design of live attenuated vaccine candidates for RSV and perhaps other pneumoviruses.