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1.
ACS Chem Biol ; 14(7): 1418-1425, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31188562

RESUMO

RNA contains diverse modifications that exert important influences in a variety of cellular processes. So far more than 150 modifications have been identified in various RNA species, mainly in rRNA and tRNA. Recent research advances in RNA modifications have been sparked by the discovery of dynamic and reversible modifications in mRNA. Moving beyond the abundant tRNA and rRNA to mRNA is opening new directions in understanding RNA modification-mediated regulation of gene expression. Recently, it was reported that N3-methylcytidine (m3C) existed in mRNA of mammalian cells, and methyltransferase-like 8 (METTL8) was identified to be the writer enzyme of m3C. However, little is known about the eraser enzyme of m3C in mRNA. In the current study, we found that the AlkB homologue 1 (ALKBH1) was capable of demethylating m3C in mRNA of mammalian cells in vitro. Overexpression and knockdown of ALKBH1 in cultured human cells can induce decrease and increase of the level of m3C in mRNA, respectively, revealing the eraser enzyme property of ALKBH1 on m3C in mRNA. In addition, we observed significant decrease of the level of m3C in mRNA in hepatocellular carcinoma (HCC) tissues compared to tumor-adjacent normal tissues, which could be attributed to the increased expression of ALKBH1 as well as the decreased expression of METTL8 in HCC tissues. These results indicated that m3C in mRNA may play certain roles in tumorigenesis. Our study shed light on understanding the demethylation of m3C in mRNA.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Citidina/análogos & derivados , RNA Mensageiro/metabolismo , Animais , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Citidina/metabolismo , Desmetilação , Células HEK293 , Humanos , Neoplasias Hepáticas/metabolismo , Mamíferos
2.
Dig Dis Sci ; 64(6): 1503-1513, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30637548

RESUMO

BACKGROUND: Reversible N6-methyladenosine (m6A) modifications in messenger RNAs can be categorized under the field of "RNA epigenetics." However, the potential role of m6A-related genes in gastric cancer (GC) prognosis has not been systematically researched. AIMS: This study was aimed at providing insights into the prognostic role of m6A-related gene expression, at both mRNA and protein levels. METHODS: Kaplan-Meier (KM) plotter database and The Cancer Genome Atlas (TCGA) database were used to explore the prognostic significance of individual m6A-related genes in overall survival (OS) and progression-free survival at the mRNA level. For independent validation, the protein level of genes significantly associated with prognosis in both databases was further detected in 450 paired GC and corresponding adjacent non-tumor tissues using tissue microarray (TMA)-based immunohistochemistry (IHC). The relationship between the FTO and ALKBH1 expression and the clinicopathological characteristics was explored. RESULTS: Among nine m6A-related genes, aberrantly high mRNA expression of FTO and ALKBH1 was associated with poor OS in the KM and TCGA cohorts. However, the TMA-IHC indicated that protein expression of FTO and ALKBH1 was markedly downregulated in GC tissues. A lower protein level of ALKBH1 was closely correlated with larger tumor sizes (≥ 5 cm) and more advanced TNM stages, while lower FTO protein expression was associated with shorter OS in GC patients. CONCLUSIONS: Aberrant expression of demethylase genes, FTO and ALKBH1, has a distinct prognostic value in GC patients, indicating that FTO and ALKBH1 may play vital roles in GC progression and metastasis.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Dioxigenase FTO Dependente de alfa-Cetoglutarato/genética , Biomarcadores Tumorais/genética , RNA Mensageiro/genética , Neoplasias Gástricas/enzimologia , Adenosina/análogos & derivados , Adenosina/metabolismo , Idoso , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Dioxigenase FTO Dependente de alfa-Cetoglutarato/metabolismo , Biomarcadores Tumorais/metabolismo , Bases de Dados Genéticas , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Processamento Pós-Transcricional do RNA , Estabilidade de RNA , RNA Mensageiro/metabolismo , Fatores de Risco , Neoplasias Gástricas/genética , Neoplasias Gástricas/mortalidade , Neoplasias Gástricas/terapia , Fatores de Tempo
3.
Nucleic Acids Res ; 46(22): 11659-11670, 2018 12 14.
Artigo em Inglês | MEDLINE | ID: mdl-30412255

RESUMO

N6-methyldeoxyadenosine (6mA) is a well-characterized DNA modification in prokaryotes but reports on its presence and function in mammals have been controversial. To address this issue, we established the capacity of 6mA-Crosslinking-Exonuclease-sequencing (6mACE-seq) to detect genome-wide 6mA at single-nucleotide-resolution, demonstrating this by accurately mapping 6mA in synthesized DNA and bacterial genomes. Using 6mACE-seq, we generated a human-genome-wide 6mA map that accurately reproduced known 6mA enrichment at active retrotransposons and revealed mitochondrial chromosome-wide 6mA clusters asymmetrically enriched on the heavy-strand. We identified a novel putative 6mA-binding protein in single-stranded DNA-binding protein 1 (SSBP1), a mitochondrial DNA (mtDNA) replication factor known to coat the heavy-strand, linking 6mA with the regulation of mtDNA replication. Finally, we characterized AlkB homologue 1 (ALKBH1) as a mitochondrial protein with 6mA demethylase activity and showed that its loss decreases mitochondrial oxidative phosphorylation. Our results show that 6mA clusters play a previously unappreciated role in regulating human mitochondrial function, despite 6mA being an uncommon DNA modification in the human genome.


Assuntos
DNA Mitocondrial/genética , Proteínas de Ligação a DNA/genética , DNA/genética , Desoxiadenosinas/genética , Genoma Mitocondrial , Proteínas Mitocondriais/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , Sequência de Bases , Mapeamento Cromossômico , DNA/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Exodesoxirribonucleases , Células HEK293 , Humanos , Proteínas Mitocondriais/metabolismo , Fosforilação Oxidativa , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Análise de Sequência de DNA , Proteínas Virais/química , Proteínas Virais/metabolismo
4.
Cell ; 175(5): 1228-1243.e20, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-30392959

RESUMO

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.


Assuntos
Adenina/análogos & derivados , Neoplasias Encefálicas/patologia , Metilação de DNA , Glioblastoma/patologia , Adenina/análise , Adenina/química , Adulto , Idoso , Homólogo AlkB 1 da Histona H2a Dioxigenase/antagonistas & inibidores , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Animais , Astrócitos/citologia , Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidade , Hipóxia Celular , Criança , Epigenômica , Feminino , Glioblastoma/metabolismo , Glioblastoma/mortalidade , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/citologia , Células-Tronco Neoplásicas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Supressora de Tumor p53/metabolismo
5.
Mol Cell ; 71(2): 306-318.e7, 2018 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-30017583

RESUMO

DNA N6-methyladenine (6mA) modification is the most prevalent DNA modification in prokaryotes, but whether it exists in human cells and whether it plays a role in human diseases remain enigmatic. Here, we showed that 6mA is extensively present in the human genome, and we cataloged 881,240 6mA sites accounting for ∼0.051% of the total adenines. [G/C]AGG[C/T] was the most significantly associated motif with 6mA modification. 6mA sites were enriched in the coding regions and mark actively transcribed genes in human cells. DNA 6mA and N6-demethyladenine modification in the human genome were mediated by methyltransferase N6AMT1 and demethylase ALKBH1, respectively. The abundance of 6mA was significantly lower in cancers, accompanied by decreased N6AMT1 and increased ALKBH1 levels, and downregulation of 6mA modification levels promoted tumorigenesis. Collectively, our results demonstrate that DNA 6mA modification is extensively present in human cells and the decrease of genomic DNA 6mA promotes human tumorigenesis.


Assuntos
Adenina/análogos & derivados , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Genoma Humano , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Adenina/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Animais , Carcinogênese/genética , DNA/genética , Metilação de DNA , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , DNA Metiltransferases Sítio Específica (Adenina-Específica)/genética
6.
Biochem Biophys Res Commun ; 495(1): 98-103, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29097205

RESUMO

Alkbh1 is a mammalian homolog of the Escherichia coli DNA repair enzyme AlkB, an Fe(II) and 2-oxoglutarate dependent dioxygenase that removes alkyl lesions from DNA bases. The human homolog ALKBH1 has been associated with six different enzymatic activities including DNA, mRNA, or tRNA hydroxylation, cleavage at abasic (AP) sites in DNA, as well as demethylation of histones. The reported cellular roles of this protein reflect the diverse enzymatic activities and include direct DNA repair, tRNA modification, and histone modification. We demonstrate that ALKBH1 produced in mammalian cells (ALKBH1293) is similar to the protein produced in bacteria (ALKBH1Ec) with regard to its m6A demethylase and AP lyase activities. In addition, we find that ALKBH1293 forms a covalent adduct with the 5' product of the lyase product in a manner analogous to ALKBH1Ec. Localization and subcellular fractionation studies with the endogenous protein in two human cell strains confirm that ALKBH1 is primarily in the mitochondria. Two strains of CRISPR/Cas9-created ALKBH1-deficient HEK293 cells showed increases in mtDNA copy number and mitochondrial dysfunction as revealed by growth measurements and citrate synthase activity assays.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Mitocôndrias/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/deficiência , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Proliferação de Células , Adutos de DNA/química , Adutos de DNA/genética , Adutos de DNA/metabolismo , Variações do Número de Cópias de DNA , DNA Mitocondrial/química , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Células HEK293 , Humanos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato
7.
Biochemistry ; 56(13): 1899-1910, 2017 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-28290676

RESUMO

Alkbh1 is one of nine mammalian homologues of Escherichia coli AlkB, a 2-oxoglutarate-dependent dioxygenase that catalyzes direct DNA repair by removing alkyl lesions from DNA. Six distinct enzymatic activities have been reported for Alkbh1, including hydroxylation of variously methylated DNA, mRNA, tRNA, or histone substrates along with the cleavage of DNA at apurinic/apyrimidinic (AP) sites followed by covalent attachment to the 5'-product. The studies described here extend the biochemical characterization of two of these enzymatic activities using human ALKBH1: the AP lyase and 6-methyl adenine DNA demethylase activities. The steady-state and single-turnover kinetic parameters for ALKBH1 cleavage of AP sites in DNA were determined and shown to be comparable to those of other AP lyases. The α,ß-unsaturated aldehyde of the 5'-product arising from DNA cleavage reacts predominantly with C129 of ALKBH1, but secondary sites also generate covalent adducts. The 6-methyl adenine demethylase activity was examined with a newly developed assay using a methylation-sensitive restriction endonuclease, and the enzymatic rate was found to be very low. Indeed, the demethylase activity was less than half that of the AP lyase activity when ALKBH1 samples were assayed using identical buffer conditions. The two enzymatic activities were examined using a series of site-directed variant proteins, revealing the presence of distinct but partially overlapping active sites for the two reactions. We postulate that the very low 6-methyl adenine oxygenase activity associated with ALKBH1 is unlikely to represent the major function of the enzyme in the cell, while the cellular role of the lyase activity (including its subsequent covalent attachment to DNA) remains uncertain.


Assuntos
Adenina/química , Homólogo AlkB 1 da Histona H2a Dioxigenase/química , DNA/química , Proteínas de Escherichia coli/química , Oxigenases de Função Mista/química , Oxirredutases O-Desmetilantes/química , Adenina/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Domínio Catalítico , DNA/genética , DNA/metabolismo , Adutos de DNA , Ensaios Enzimáticos , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Humanos , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Modelos Moleculares , Oligonucleotídeos/química , Oligonucleotídeos/metabolismo , Oxirredutases O-Desmetilantes/genética , Oxirredutases O-Desmetilantes/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade da Espécie
8.
Cell ; 167(3): 816-828.e16, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27745969

RESUMO

tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N1-methyladenosine (m1A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation.


Assuntos
Homólogo AlkB 1 da Histona H2a Dioxigenase/metabolismo , Regulação da Expressão Gênica , Biossíntese de Proteínas/genética , RNA de Transferência/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Homólogo AlkB 1 da Histona H2a Dioxigenase/genética , Glucose/deficiência , Células HeLa , Humanos , Metilação , Polirribossomos/metabolismo
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