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1.
Aging (Albany NY) ; 13(12): 16287-16315, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34230220

RESUMO

N6-methyladenosine (m6A) RNA methylation is associated with malignant tumor progression and is modulated by various m6A RNA methylation regulator proteins. However, its role in endometrial cancer is unclear. In this work, we analyzed sequence, copy number variation, and clinical data obtained from the TCGA database. Expression was validated using real-time quantitative polymerase chain reaction and immunohistochemistry. Changes in m6A RNA methylation regulators were closely related to the clinicopathological stage and prognosis of endometrial cancer. In particular, ZC3H13, YTHDC1, and METTL14 were identified as potential markers for endometrial cancer diagnosis and prognosis. The TIMER algorithm indicated that immune cell infiltration correlated with changes in ZC3H13, YTHDC1, and METTL14 expression. Meanwhile, ZC3H13 or YTHDC1 knockdown promoted the proliferation and invasion of endometrial cancer cells. Through gene enrichment analysis, we constructed a regulatory network in order to explore the potential molecular mechanism involving ZC3H13, YTHDC1, and METTL14. Virtual screening predicted interactions of potential therapeutic compounds with METTL14 and YTHDC1. These findings advance the understanding of RNA epigenetic modifications in endometrial cancer while identifying m6A regulators associated with immune infiltration, prognosis, and potential treatment strategies.


Assuntos
Adenosina/análogos & derivados , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/imunologia , Linfócitos do Interstício Tumoral/imunologia , Adenosina/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Variações do Número de Cópias de DNA/genética , Intervalo Livre de Doença , Neoplasias do Endométrio/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Ligantes , Metilação , Pessoa de Meia-Idade , Simulação de Acoplamento Molecular , Mutação/genética , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Prognóstico , Modelos de Riscos Proporcionais , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Microambiente Tumoral/imunologia
2.
Nat Commun ; 12(1): 3780, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145242

RESUMO

RNA N6-methyladenosine (m6A), the most abundant internal modification of mRNAs, plays key roles in human development and health. Post-translational methylation of proteins is often critical for the dynamic regulation of enzymatic activity. However, the role of methylation of the core methyltransferase METTL3/METTL14 in m6A regulation remains elusive. We find by mass spectrometry that METTL14 arginine 255 (R255) is methylated (R255me). Global mRNA m6A levels are greatly decreased in METTL14 R255K mutant mouse embryonic stem cells (mESCs). We further find that R255me greatly enhances the interaction of METTL3/METTL14 with WTAP and promotes the binding of the complex to substrate RNA. We show that protein arginine N-methyltransferases 1 (PRMT1) interacts with and methylates METTL14 at R255, and consistent with this, loss of PRMT1 reduces mRNA m6A modification globally. Lastly, we find that loss of R255me preferentially affects endoderm differentiation in mESCs. Collectively, our findings show that arginine methylation of METTL14 stabilizes the binding of the m6A methyltransferase complex to its substrate RNA, thereby promoting global m6A modification and mESC endoderm differentiation. This work highlights the crosstalk between protein methylation and RNA methylation in gene expression.


Assuntos
Adenosina/análogos & derivados , Arginina/metabolismo , Endoderma/citologia , Metiltransferases/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Adenosina/genética , Adenosina/metabolismo , Animais , Diferenciação Celular/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Camundongos , Processamento de Proteína Pós-Traducional/genética , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/genética
3.
Nat Commun ; 12(1): 3778, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34145251

RESUMO

N6-methyladenosine (m6A) is the most abundant internal modification on mRNA which influences most steps of mRNA metabolism and is involved in several biological functions. The E3 ubiquitin ligase Hakai was previously found in complex with components of the m6A methylation machinery in plants and mammalian cells but its precise function remained to be investigated. Here we show that Hakai is a conserved component of the methyltransferase complex in Drosophila and human cells. In Drosophila, its depletion results in reduced m6A levels and altered m6A-dependent functions including sex determination. We show that its ubiquitination domain is required for dimerization and interaction with other members of the m6A machinery, while its catalytic activity is dispensable. Finally, we demonstrate that the loss of Hakai destabilizes several subunits of the methyltransferase complex, resulting in impaired m6A deposition. Our work adds functional and molecular insights into the mechanism of the m6A mRNA writer complex.


Assuntos
Adenosina/análogos & derivados , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Metiltransferases/metabolismo , RNA Mensageiro/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Adenosina/metabolismo , Animais , Linhagem Celular , Drosophila melanogaster , Células HeLa , Humanos , Metilação , Metiltransferases/genética , Processamento Pós-Transcricional do RNA/genética , Splicing de RNA/genética
4.
J Transl Med ; 19(1): 251, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103054

RESUMO

N6-methyl-adenosine (m6A) is one of the most common internal modifications on RNA molecules present in mammalian cells. Deregulation of m6A modification has been recently implicated in many types of human diseases. Therefore, m6A modification has become a research hotspot for its potential therapeutic applications in the treatment of various diseases. The immune system mostly involves different types of immune cells to provide the first line of defense against infections. The immunoregulatory network that orchestrate the immune responses to new pathogens plays a pivotal role in the development of the disease. And m6A modification has been demonstrated to be a major post-transcriptional regulator of immune responses in cells. In this review, we summarize the participants involved in m6A regulation and try to reveal how m6A modification affects the immune responses via changing the immunoregulatory networks.


Assuntos
Adenosina , Adenosina/análogos & derivados , Animais , Humanos
5.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: covidwho-1281915

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
6.
Nat Commun ; 12(1): 3803, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155197

RESUMO

The adenomatous polyposis coli (APC) is a frequently mutated tumour suppressor gene in cancers. However, whether APC is regulated at the epitranscriptomic level remains elusive. In this study, we analysed TCGA data and separated 200 paired oesophageal squamous cell carcinoma (ESCC) specimens and their adjacent normal tissues and demonstrated that methyltransferase-like 3 (METTL3) is highly expressed in tumour tissues. m6A-RNA immunoprecipitation sequencing revealed that METTL3 upregulates the m6A modification of APC, which recruits YTHDF for APC mRNA degradation. Reduced APC expression increases the expression of ß-catenin and ß-catenin-mediated cyclin D1, c-Myc, and PKM2 expression, thereby leading to enhanced aerobic glycolysis, ESCC cell proliferation, and tumour formation in mice. In addition, downregulated APC expression correlates with upregulated METTL3 expression in human ESCC specimens and poor prognosis in ESCC patients. Our findings reveal a mechanism by which the Wnt/ß-catenin pathway is upregulated in ESCC via METTL3/YTHDF-coupled epitranscriptomal downregulation of APC.


Assuntos
Adenosina/análogos & derivados , Proteínas do Citoesqueleto/genética , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/metabolismo , Animais , Carcinogênese , Proliferação de Células , Proteínas do Citoesqueleto/metabolismo , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Humanos , Metiltransferases/genética , Camundongos , Prognóstico , RNA Mensageiro/metabolismo , Efeito Warburg em Oncologia , Via de Sinalização Wnt , beta Catenina/genética , beta Catenina/metabolismo
7.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
8.
Plant Cell Rep ; 40(7): 1229-1245, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34081180

RESUMO

KEY MESSAGE: 9 YTH genes in tomato were identified and cloned, and their expression patterns were comprehensively analyzed, which reveal potential multiple roles in development and fruit ripening. N6-methyladenosine (m6A) is an abundant and pervasive post-transcriptional modification in eukaryotic mRNAs. The YTH domain-containing proteins act as m6A readers to read m6A marks and transduce their downstream regulatory effects by altering m6A-mRNA metabolism processes. Identification of YTH proteins is essential for understanding the regulatory mechanisms of m6A in physiological processes, but little is known about YTH proteins in tomato, a model system for fruit development. Here, we report that tomato genomes contain a total of 9 SlYTH genes. While YTH proteins of both tomato and Arabidopsis can be classified into two subfamilies, the member distributions in subfamilies are very different between the two species. Homology modeling exhibited the similar three-dimensional structures of SlYTH proteins to human YTHDF1 or YTHDC1. Multiple hormone-response elements locating on the promoters of SlYTH genes indicate that they are involved in the physiological processes related to phytohormone. SlYTH genes are ubiquitous and spatiotemporal dynamic expression in tomato. Eight SlTYH genes have the strongest expression in stamens among the parts of flowers. Throughout fruit ontogeny, most of the SlYTH genes display obvious high mRNA levels during the developmental phases (4 dpa to mature green); moreover, SlYTH1 and SlYTH2 have absolute predominant expressions demonstrated by RNA-seq. The results lay a foundation for future characterizations on the functions of YTH proteins and m6A regulatory mechanism in tomato.


Assuntos
Adenosina/análogos & derivados , Lycopersicon esculentum/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Adenosina/genética , Mapeamento Cromossômico , Flores/genética , Flores/crescimento & desenvolvimento , Frutas/genética , Frutas/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Ácidos Indolacéticos/metabolismo , Ácidos Indolacéticos/farmacologia , Lycopersicon esculentum/efeitos dos fármacos , Lycopersicon esculentum/crescimento & desenvolvimento , Família Multigênica , Filogenia , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Domínios Proteicos , Proteínas de Ligação a RNA/química , Homologia Estrutural de Proteína , Sintenia
9.
Molecules ; 26(10)2021 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067988

RESUMO

A carboxylated adenosine analog (C-Ado-) has been synthesized and probed via time-resolved photoelectron spectroscopy in order to induce intra-molecular charge transfer from the carboxylic acid moiety to the nucleobase. Intra-molecular charge transfer can be exploited as starting point to probe low-energy electron (LEE) damage in DNA and its derivatives. Time-dependent density functional theory (TD-DFT) calculations at the B3LYP-6311G level of theory have been performed to verify that the highest occupied molecular orbital (HOMO) was located on carboxylic acid and that the lowest occupied molecular orbital (LUMO) was on the nucleobase. Hence, the carboxylic acid could work as electron source, whilst the nucleobase could serve the purpose of electron acceptor. The dynamics following excitation at 4.66 eV (266 nm) were probed using time-resolved photoelectron spectroscopy using probes at 1.55 eV (800 nm) and 3.10 eV (400 nm). The data show rapid decay of the excited state population and, based on the similarity of the overall dynamics to deoxy-adenosine monophosphate (dAMP-), it appears that the dominant decay mechanism is internal conversion following 1ππ* excitation of the nucleobase, rather than charge-transfer from the carboxylic acid to the nucleobase.


Assuntos
Adenosina/análogos & derivados , Adenosina/química , Elétrons , Luz , Modelos Moleculares , Conformação Molecular , Oxirredução/efeitos da radiação , Termodinâmica , Fatores de Tempo
10.
Methods Mol Biol ; 2298: 399-414, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085257

RESUMO

N6-methyladenosine (m6A) is a major epitranscriptomic mark exerting crucial diverse roles in RNA metabolisms, including RNA stability, mRNA translation, and RNA structural rearrangement. m6A modifications at different RNA regions may have distinct molecular effects. Here, we describe a CRISPR-Cas9-based approach that enables targeted m6A addition or removal on endogenous RNA molecules without altering the nucleotide sequence. By fusing a catalytically inactive Cas9 with engineered m6A modification enzymes, the programmable m6A editors are capable of achieving RNA methylation and demethylation at desired sites, facilitating dissection of regional effects of m6A and diversifying the toolkits for RNA manipulation.


Assuntos
Adenosina/análogos & derivados , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , RNA/genética , Adenosina/genética , Sequência de Bases/genética , Linhagem Celular Tumoral , Edição de Genes/métodos , Células HeLa , Humanos , Metilação , RNA Mensageiro/genética
11.
Medicine (Baltimore) ; 100(20): e25952, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34011074

RESUMO

BACKGROUND: Osteosarcoma represents the most common malignant bone tumor with high metastatic potential and inferior prognosis. RNA methylation (N6-methyladenosine [m6A]) is a prevalent RNA modification that epigenetically influences numerous biological processes including tumorigenesis. This study aims to determine that m6A regulators are significant biomarkers for osteosarcoma, and establish a prognostic model to predict the survival of patients. METHODS: In this study, we comprehensively analyzed the underlying associations between m6A regulators' mRNA expressions and metastasis as well as prognosis of osteosarcoma patients in the Cancer Genome Atlas. Multivariate Cox-regression analysis was used to screen regulators that were significantly associated with overall survival of osteosarcoma patients. Least absolute shrinkage and selection operator (LASSO) Cox-regression analysis was used for constructing m6A regulator-based osteosarcoma prognostic signature. RESULTS: Some of the regulators exhibited aberrant mRNA levels between osteosarcoma samples with and without metastasis. Multivariate Cox-regression analysis identified several regulators with potential prognostic significance. A risk score formula consisted of methyltransferase-like 3, YTH domains of Homo sapiens, and fat mass and obesity-associated protein was obtained through which patients could be prognostically stratified independently of potential confounding factors. The signature was also significantly associated with the metastatic potential of osteosarcoma. All the analyses could be well reproduced in another independent osteosarcoma cohort from the Gene Expression Omnibus. CONCLUSIONS: In conclusion, this study first revealed potential roles of m6A regulators in osteosarcoma metastasis and prognosis, which should be helpful for its clinical decision-making.


Assuntos
Biomarcadores Tumorais/genética , Osteossarcoma/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/análise , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Conjuntos de Dados como Assunto , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Metilação , Metiltransferases/análise , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteossarcoma/mortalidade , Osteossarcoma/secundário , Prognóstico , Modelos de Riscos Proporcionais , Fatores de Processamento de RNA/análise , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , RNA-Seq
12.
Nat Commun ; 12(1): 3244, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050143

RESUMO

N6-methyladenosine (m6A) is a modification that plays pivotal roles in RNA metabolism and function, although its functions in spliceosomal U6 snRNA remain unknown. To elucidate its role, we conduct a large-scale transcriptome analysis of a Schizosaccharomyces pombe strain lacking this modification and found a global change of pre-mRNA splicing. The most significantly impacted introns are enriched for adenosine at the fourth position pairing the m6A in U6 snRNA, and exon sequences weakly recognized by U5 snRNA. This suggests cooperative recognition of 5' splice site by U6 and U5 snRNPs, and also a role of m6A facilitating efficient recognition of the splice sites weakly interacting with U5 snRNA, indicating that U6 snRNA m6A relaxes the 5' exon constraint and allows protein sequence diversity along with explosively increasing number of introns over the course of eukaryotic evolution.


Assuntos
Regulação Fúngica da Expressão Gênica , Splicing de RNA , RNA Fúngico/metabolismo , RNA Nuclear Pequeno/metabolismo , Schizosaccharomyces/genética , Regiões 3' não Traduzidas/genética , Regiões 5' não Traduzidas/genética , Adenosina/análogos & derivados , Adenosina/metabolismo , Éxons/genética , Espectrometria de Massas , Metiltransferases/genética , Metiltransferases/isolamento & purificação , Metiltransferases/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA/genética , RNA Fúngico/genética , RNA Nuclear Pequeno/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U4-U6 , Ribonucleoproteína Nuclear Pequena U5 , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/isolamento & purificação , Proteínas de Schizosaccharomyces pombe/metabolismo
14.
Int J Mol Sci ; 22(9)2021 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-33946178

RESUMO

Growth and maturation of hematopoietic stem cells (HSCs) are largely controlled at both transcriptional and post-transcriptional levels. In particular, hematopoietic development requires a tight control of protein synthesis. Furthermore, translational deregulation strongly contributes to hematopoietic malignancies. Researchers have recently identified a new layer of gene expression regulation that consists of chemical modification of RNA species, which led to the birth of the epitranscriptomics field. RNA modifications provide an additional level of control in hematopoietic development by acting as post-transcriptional regulators of lineage-specific genetic programs. Other reviews have already described the important role of the N6-methylation of adenosine (m6A) within mRNA species in regulating hematopoietic differentiation and diseases. The aim of this review is to summarize the current status of the role of RNA modifications in the regulation of ribosome function, beyond m6A. In particular, we discuss the importance of RNA modifications in tRNA and rRNA molecules. By balancing translational rate and fidelity, they play an important role in regulating normal and malignant hematopoietic development.


Assuntos
Adenosina/análogos & derivados , Leucemia/genética , Processamento Pós-Transcricional do RNA , RNA/genética , Ribossomos/genética , Adenosina/genética , Animais , Regulação Leucêmica da Expressão Gênica , Humanos , RNA Mensageiro/genética , RNA Ribossômico/genética , RNA de Transferência/genética
15.
Nucleic Acids Res ; 49(10): 5568-5587, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33999208

RESUMO

Heterochromatin has essential functions in maintaining chromosome structure, in protecting genome integrity and in stabilizing gene expression programs. Heterochromatin is often nucleated by underlying DNA repeat sequences, such as major satellite repeats (MSR) and long interspersed nuclear elements (LINE). In order to establish heterochromatin, MSR and LINE elements need to be transcriptionally competent and generate non-coding repeat RNA that remain chromatin associated. We explored whether these heterochromatic RNA, similar to DNA and histones, may be methylated, particularly for 5-methylcytosine (5mC) or methyl-6-adenosine (m6A). Our analysis in mouse ES cells identifies only background level of 5mC but significant enrichment for m6A on heterochromatic RNA. Moreover, MSR transcripts are a novel target for m6A RNA modification, and their m6A RNA enrichment is decreased in ES cells that are mutant for Mettl3 or Mettl14, which encode components of a central RNA methyltransferase complex. Importantly, MSR transcripts that are partially deficient in m6A RNA methylation display impaired chromatin association and have a reduced potential to form RNA:DNA hybrids. We propose that m6A modification of MSR RNA will enhance the functions of MSR repeat transcripts to stabilize mouse heterochromatin.


Assuntos
DNA/metabolismo , Heterocromatina , RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Metilação , Camundongos , Células-Tronco Embrionárias Murinas , Sequências de Repetição em Tandem
16.
BMC Genomics ; 22(1): 296, 2021 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-33888086

RESUMO

BACKGROUND: The newly discovered reversible N6-methyladenosine (m6A) modification plays an important regulatory role in gene expression. Long non-coding RNAs (lncRNAs) participate in Marek's disease virus (MDV) replication but how m6A modifications in lncRNAs are affected during MDV infection is currently unknown. Herein, we profiled the transcriptome-wide m6A modification in lncRNAs in MDV-infected chicken embryo fibroblast (CEF) cells. RESULTS: Methylated RNA immunoprecipitation sequencing results revealed that the lncRNA m6A modification is highly conserved with MDV infection increasing the expression of lncRNA m6A modified sites compared to uninfected cell controls. Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that lncRNA m6A modifications were highly associated with signaling pathways associated with MDV infection. CONCLUSIONS: In this study, the alterations seen in transcriptome-wide m6A occurring in lncRNAs following MDV-infection suggest this process plays important regulatory roles during MDV replication. We report for the first time profiling of the alterations in transcriptome-wide m6A modification in lncRNAs of MDV-infected CEF cells.


Assuntos
Herpesvirus Galináceo 2 , Doença de Marek , RNA Longo não Codificante , Adenosina/análogos & derivados , Animais , Embrião de Galinha , Galinhas/genética , Doença de Marek/genética , RNA Longo não Codificante/genética , Transcriptoma , Replicação Viral
17.
Nat Commun ; 12(1): 2183, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33846348

RESUMO

Here we show that FTO as an N6-methyladenosine (m6A) RNA demethylase is degraded by selective autophagy, which is impaired by low-level arsenic exposure to promote tumorigenesis. We found that in arsenic-associated human skin lesions, FTO is upregulated, while m6A RNA methylation is downregulated. In keratinocytes, chronic relevant low-level arsenic exposure upregulated FTO, downregulated m6A RNA methylation, and induced malignant transformation and tumorigenesis. FTO deletion inhibited arsenic-induced tumorigenesis. Moreover, in mice, epidermis-specific FTO deletion prevented skin tumorigenesis induced by arsenic and UVB irradiation. Targeting FTO genetically or pharmacologically inhibits the tumorigenicity of arsenic-transformed tumor cells. We identified NEDD4L as the m6A-modified gene target of FTO. Finally, arsenic stabilizes FTO protein through inhibiting p62-mediated selective autophagy. FTO upregulation can in turn inhibit autophagy, leading to a positive feedback loop to maintain FTO accumulation. Our study reveals FTO-mediated dysregulation of mRNA m6A methylation as an epitranscriptomic mechanism to promote arsenic tumorigenicity.


Assuntos
Adenosina/análogos & derivados , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Arsênio/toxicidade , Autofagia , Carcinogênese/genética , Adenosina/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Animais , Autofagia/efeitos dos fármacos , Autofagia/genética , Sequência de Bases , Carcinogênese/efeitos dos fármacos , Transformação Celular Neoplásica/efeitos dos fármacos , Transformação Celular Neoplásica/patologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Epiderme/metabolismo , Ontologia Genética , Células HEK293 , Células HaCaT , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Camundongos , NF-kappa B/metabolismo , Ubiquitina-Proteína Ligases Nedd4/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Estabilidade de RNA/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteína Sequestossoma-1/metabolismo , Transcriptoma/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
18.
Antimicrob Agents Chemother ; 65(7): e0256620, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-33875421

RESUMO

Chikungunya virus (CHIKV) nonstructural protein 1 (nsP1) harbors the methyltransferase (MTase) and guanylyltransferase (GTase) activities needed for viral RNA capping and represents a promising antiviral drug target. We compared the antiviral efficacies of nsP1 inhibitors belonging to the MADTP, CHVB, and FHNA series (6'-fluoro-homoneplanocin A [FHNA], its 3'-keto form, and 6'-ß-fluoro-homoaristeromycin). Cell-based phenotypic cross-resistance assays revealed that the CHVB and MADTP series had similar modes of action that differed from that of the FHNA series. In biochemical assays with purified Semliki Forest virus and CHIKV nsP1, CHVB compounds strongly inhibited MTase and GTase activities, while MADTP-372 had a moderate inhibitory effect. FHNA did not directly inhibit the enzymatic activity of CHIKV nsP1. The first-of-their-kind molecular-docking studies with the cryo-electron microscopy (cryo-EM) structure of CHIKV nsP1, which is assembled into a dodecameric ring, revealed that the MADTP and CHVB series bind at the S-adenosylmethionine (SAM)-binding site in the capping domain, where they would function as competitive or noncompetitive inhibitors. The FHNA series was predicted to bind at the secondary binding pocket in the ring-aperture membrane-binding and oligomerization (RAMBO) domain, potentially interfering with the membrane binding and oligomerization of nsP1. Our cell-based and enzymatic assays, in combination with molecular docking and mapping of compound resistance mutations to the nsP1 structure, allowed us to group nsP1 inhibitors into functionally distinct classes. This study identified druggable pockets in the nsP1 dodecameric structure and provides a basis for the rational design, optimization, and combination of inhibitors of this unique and promising antiviral drug target.


Assuntos
Vírus Chikungunya , Proteínas não Estruturais Virais , Adenosina/análogos & derivados , Microscopia Crioeletrônica , Simulação de Acoplamento Molecular , Proteínas não Estruturais Virais/genética , Replicação Viral
19.
SLAS Discov ; 26(6): 757-765, 2021 07.
Artigo em Inglês | MEDLINE | ID: covidwho-1194439

RESUMO

Frequent outbreaks of novel coronaviruses (CoVs), highlighted by the current severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, necessitate the development of therapeutics that could be easily and effectively administered worldwide. The conserved mRNA-capping process enables CoVs to evade their host immune system and is a target for antiviral development. Nonstructural protein (nsp) 16 in complex with nsp10 catalyzes the final step of coronaviral mRNA capping through its 2'-O-methylation activity. Like other methyltransferases, the SARS-CoV-2 nsp10-nsp16 complex is druggable. However, the availability of an optimized assay for high-throughput screening (HTS) is an unmet need. Here, we report the development of a radioactivity-based assay for the methyltransferase activity of the nsp10-nsp16 complex in a 384-well format, kinetic characterization, and optimization of the assay for HTS (Z' factor = 0.83). Considering the high conservation of nsp16 across known CoV species, the potential inhibitors targeting the SARS-CoV-2 nsp10-nsp16 complex may also be effective against other emerging pathogenic CoVs.


Assuntos
Adenosina/análogos & derivados , Ensaios de Triagem em Larga Escala , Capuzes de RNA/antagonistas & inibidores , RNA Viral/antagonistas & inibidores , Proteínas não Estruturais Virais/antagonistas & inibidores , Proteínas Virais Reguladoras e Acessórias/antagonistas & inibidores , Adenosina/química , Adenosina/farmacologia , COVID-19/virologia , Clonagem Molecular , Ensaios Enzimáticos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Metilação , Modelos Moleculares , Capuzes de RNA/genética , Capuzes de RNA/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SARS-CoV-2/efeitos dos fármacos , SARS-CoV-2/enzimologia , SARS-CoV-2/genética , Trítio , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genética , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais Reguladoras e Acessórias/química , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
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