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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
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.
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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
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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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
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.
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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
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.
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Adenosina/análogos & derivados , Infecções por Vírus Respiratório Sincicial/imunologia , Vacinas contra Vírus Sincicial Respiratório/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Replicação Viral/imunologia , Células A549 , Adenosina/genética , Adenosina/imunologia , Adenosina/metabolismo , Animais , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Feminino , Células HeLa , Humanos , Masculino , Infecções por Vírus Respiratório Sincicial/virologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/patogenicidade , Sigmodontinae , Regulação para Cima/imunologia , Vacinas Atenuadas/imunologia , Células Vero , Virulência/genética , Virulência/imunologia , Replicação Viral/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.
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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 Figure 5, translation initiation is promoted not by the indicated protein, but by YTHDF1 (see below).
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In the version of this Article originally published, the authors incorrectly listed an accession code as GES90642. The correct code is GSE90642 . This has now been amended in all online versions of the Article.
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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
N6-methyladenosine (m6A) is the most prevalent modification in eukaryotic messenger RNAs (mRNAs) and is interpreted by its readers, such as YTH domain-containing proteins, to regulate mRNA fate. Here, we report the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; including IGF2BP1/2/3) as a distinct family of m6A readers that target thousands of mRNA transcripts through recognizing the consensus GG(m6A)C sequence. In contrast to the mRNA-decay-promoting function of YTH domain-containing family protein 2, IGF2BPs promote the stability and storage of their target mRNAs (for example, MYC) in an m6A-dependent manner under normal and stress conditions and therefore affect gene expression output. Moreover, the K homology domains of IGF2BPs are required for their recognition of m6A and are critical for their oncogenic functions. Thus, our work reveals a different facet of the m6A-reading process that promotes mRNA stability and translation, and highlights the functional importance of IGF2BPs as m6A readers in post-transcriptional gene regulation and cancer biology.
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Adenosina/análogos & derivados , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/genética , Adenosina/metabolismo , Sítios de Ligação , Movimento Celular , Proliferação de Células , Sequência Consenso , Feminino , Sangue Fetal/citologia , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Células HeLa , Células-Tronco Hematopoéticas/enzimologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Invasividade Neoplásica , Ligação Proteica , Biossíntese de Proteínas , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologiaRESUMO
N6-methyladenosine (m6A), the most prevalent internal modification in eukaryotic messenger RNAs (mRNAs), plays critical roles in many bioprocesses. However, its functions in normal and malignant hematopoiesis remain elusive. Here, we report that METTL14, a key component of the m6A methyltransferase complex, is highly expressed in normal hematopoietic stem/progenitor cells (HSPCs) and acute myeloid leukemia (AML) cells carrying t(11q23), t(15;17), or t(8;21) and is downregulated during myeloid differentiation. Silencing of METTL14 promotes terminal myeloid differentiation of normal HSPCs and AML cells and inhibits AML cell survival/proliferation. METTL14 is required for development and maintenance of AML and self-renewal of leukemia stem/initiation cells (LSCs/LICs). Mechanistically, METTL14 exerts its oncogenic role by regulating its mRNA targets (e.g., MYB and MYC) through m6A modification, while the protein itself is negatively regulated by SPI1. Collectively, our results reveal the SPI1-METTL14-MYB/MYC signaling axis in myelopoiesis and leukemogenesis and highlight the critical roles of METTL14 and m6A modification in normal and malignant hematopoiesis.
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Adenosina/análogos & derivados , Carcinogênese/genética , Carcinogênese/patologia , Diferenciação Celular , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/patologia , Metiltransferases/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese/metabolismo , Proliferação de Células , Sobrevivência Celular , Regulação para Baixo/genética , Regulação Leucêmica da Expressão Gênica , Células HEK293 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Metiltransferases/genética , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Biossíntese de Proteínas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-myb/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transativadores/metabolismo , Transcriptoma/genética , Regulação para Cima/genéticaAssuntos
Precursores de RNA , RNA , Adenosina/análogos & derivados , Adenosina/química , Éxons , Metilação , RNA Mensageiro/químicaRESUMO
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.
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Células Germinativas , Zigoto , Animais , Feminino , Fertilidade , Humanos , Oócitos , OogêneseRESUMO
The dynamic and reversible N6-methyladenosine (m6A) RNA modification installed and erased by N6-methyltransferases and demethylases regulates gene expression and cell fate. We show that the m6A demethylase ALKBH5 is highly expressed in glioblastoma stem-like cells (GSCs). Silencing ALKBH5 suppresses the proliferation of patient-derived GSCs. Integrated transcriptome and m6A-seq analyses revealed altered expression of certain ALKBH5 target genes, including the transcription factor FOXM1. ALKBH5 demethylates FOXM1 nascent transcripts, leading to enhanced FOXM1 expression. Furthermore, a long non-coding RNA antisense to FOXM1 (FOXM1-AS) promotes the interaction of ALKBH5 with FOXM1 nascent transcripts. Depleting ALKBH5 and FOXM1-AS disrupted GSC tumorigenesis through the FOXM1 axis. Our work uncovers a critical function for ALKBH5 and provides insight into critical roles of m6A methylation in glioblastoma.
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Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Neoplasias Encefálicas/patologia , Proteína Forkhead Box M1/metabolismo , Glioblastoma/patologia , Regiões 3' não Traduzidas , Homólogo AlkB 5 da RNA Desmetilase/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/mortalidade , Linhagem Celular Tumoral , Proliferação de Células , Proteína Semelhante a ELAV 1/genética , Proteína Semelhante a ELAV 1/metabolismo , Proteína Forkhead Box M1/genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/mortalidade , Masculino , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
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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
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.
