共查询到20条相似文献,搜索用时 93 毫秒
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《Biochemical and biophysical research communications》2020,521(2):499-506
Obstructive sleep apnea (OSA) is closely associated with cancer progression and cancer-related mortality. N6-methyladenosine (m6A) is involved in the process of intermittent hypoxia (IH) promoting tumor progression. However, it is unclear how m6A regulates the development of lung adenocarcinoma under IH. In this study, we found that ALKBH5 was elevated in lung adenocarcinoma cells and subcutaneous tumors in mice under IH, which was associated with decreased m6A levels in these cells and tissues. Next, we knocked out ALKBH5 in a human lung adenocarcinoma cell line under IH, and we found that the proliferation and invasion of these cells were significantly inhibited. Mechanistic analysis showed that under IH, knockout of ALKBH5 in lung adenocarcinoma cells upregulated the level of m6A in Forkhead box M1 (FOXM1) mRNA and decreased the translation efficiency of FOXM1 mRNA, resulting in downregulation of the FOXM1 protein. The FOXM1 protein is elevated in lung adenocarcinoma cells and subcutaneous tumor tissues of mice under IH. By knocking out FOXM1 in lung adenocarcinoma cells under IH, proliferation and invasion of these cells were inhibited, and overexpression of FOXM1 partially restored the inhibition of growth and invasion of lung adenocarcinoma cells due to ALKBH5 knockout. Collectively, our findings demonstrate that the m6A demethylase ALKBH5 affects the proliferation and invasion of lung adenocarcinoma cells under IH by downregulating m6A modification on FOXM1 mRNA and by promoting FOXM1 expression. 相似文献
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Tingting Yu Lan Yao Hua Yin Yao Teng Mei Hong Qiuling Wu 《International journal of biological sciences》2022,18(6):2235
N6-methyladenosine (m6A) is the most prevalent modification to RNA in higher eukaryotes. ALKBH5 is an RNA demethylase that impacts RNA export and metabolism, and its aberrant expression is associated with the generation of tumours. In this study, we found that ALKBH5 was highly expressed in both primary CD138+ plasma cells isolated from multiple myeloma (MM) patients and MM cell lines. Downregulation of ALKBH5 inhibited myeloma cell proliferation, neovascularization, invasion and migration ability, and promoted the apoptosis in vivo and in vitro. MeRIP-seq identified the SAV1 gene as main target gene of ALKBH5. Inhibiting ALKBH5 in MM cells increased SAV1 m6A levels, decreased SAV1 mRNA stability and expression, suppressed the stem cell related HIPPO-pathway signalling and ultimately activates the downstream effector YAP, exerting an anti-myeloma effect. Additionally, MM stem cell phenotype was suppressed in ALKBH5-deficient cells and the expression of pluripotency factors NANOG, SOX2 and OCT4 were also decreased. Altogether, our results suggest that ALKBH5 acts as an oncogene in MM and might serve as an attractive potential biomarker and therapeutic target. 相似文献
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Chao Xu Ke Liu Wolfram Tempel Marina Demetriades WeiShen Aik Christopher J. Schofield Jinrong Min 《The Journal of biological chemistry》2014,289(25):17299-17311
N6-Methyladenosine (m6A) is the most prevalent internal RNA modification in eukaryotes. ALKBH5 belongs to the AlkB family of dioxygenases and has been shown to specifically demethylate m6A in single-stranded RNA. Here we report crystal structures of ALKBH5 in the presence of either its cofactors or the ALKBH5 inhibitor citrate. Catalytic assays demonstrate that the ALKBH5 catalytic domain can demethylate both single-stranded RNA and single-stranded DNA. We identify the TCA cycle intermediate citrate as a modest inhibitor of ALKHB5 (IC50, ∼488 μm). The structural analysis reveals that a loop region of ALKBH5 is immobilized by a disulfide bond that apparently excludes the binding of dsDNA to ALKBH5. We identify the m6A binding pocket of ALKBH5 and the key residues involved in m6A recognition using mutagenesis and ITC binding experiments. 相似文献
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Fang Yu Jiangbo Wei Xiaolong Cui Chunjie Yu Wei Ni Jrg Bungert Lizi Wu Chuan He Zhijian Qian 《Nucleic acids research》2021,49(10):5779
Faithful genome integrity maintenance plays an essential role in cell survival. Here, we identify the RNA demethylase ALKBH5 as a key regulator that protects cells from DNA damage and apoptosis during reactive oxygen species (ROS)-induced stress. We find that ROS significantly induces global mRNA N6-methyladenosine (m6A) levels by modulating ALKBH5 post-translational modifications (PTMs), leading to the rapid and efficient induction of thousands of genes involved in a variety of biological processes including DNA damage repair. Mechanistically, ROS promotes ALKBH5 SUMOylation through activating ERK/JNK signaling, leading to inhibition of ALKBH5 m6A demethylase activity by blocking substrate accessibility. Moreover, ERK/JNK/ALKBH5-PTMs/m6A axis is activated by ROS in hematopoietic stem/progenitor cells (HSPCs) in vivo in mice, suggesting a physiological role of this molecular pathway in the maintenance of genome stability in HSPCs. Together, our study uncovers a molecular mechanism involving ALKBH5 PTMs and increased mRNA m6A levels that protect genomic integrity of cells in response to ROS. 相似文献
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Weizhong Chen Liang Zhang Guanqun Zheng Ye Fu Quanjiang Ji Fange Liu Hao Chen Chuan He 《FEBS letters》2014
ALKBH5, a member of AlkB family proteins, has been reported as a mammalian N6-methyladenosine (m6A) RNA demethylase. Here we report the crystal structure of zebrafish ALKBH5 (fALKBH5) with the resolution of 1.65 Å. Structural superimposition shows that fALKBH5 is comprised of a conserved jelly-roll motif. However, it possesses a loop that interferes potential binding of a duplex nucleic acid substrate, suggesting an important role in substrate selection. In addition, several active site residues are different between the two known m6A RNA demethylases, ALKBH5 and FTO, which may result in their slightly different pathways of m6A demethylation. 相似文献
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Yue Huang Jingli Yan Qi Li Jiafei Li Shouzhe Gong Hu Zhou Jianhua Gan Hualiang Jiang Gui-Fang Jia Cheng Luo Cai-Guang Yang 《Nucleic acids research》2015,43(1):373-384
Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N6-methyladenosine (m6A) residues in mRNA. Achieving a method for selective inhibition of FTO over ALKBH5 remains a challenge, however. Here, we have identified meclofenamic acid (MA) as a highly selective inhibitor of FTO. MA is a non-steroidal, anti-inflammatory drug that mechanistic studies indicate competes with FTO binding for the m6A-containing nucleic acid. The structure of FTO/MA has revealed much about the inhibitory function of FTO. Our newfound understanding, revealed herein, of the part of the nucleotide recognition lid (NRL) in FTO, for example, has helped elucidate the principles behind the selectivity of FTO over ALKBH5. Treatment of HeLa cells with the ethyl ester form of MA (MA2) has led to elevated levels of m6A modification in mRNA. Our collective results highlight the development of functional probes of the FTO enzyme that will (i) enable future biological studies and (ii) pave the way for the rational design of potent and specific inhibitors of FTO for use in medicine. 相似文献
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《Reproductive biology》2022,22(1):100611
Long non-coding RNAs (lncRNAs) have been elucidated to play vital roles in the phenotype of trophoblast cells. Nevertheless, the effect of SNHG1 has not been investigated on trophoblast cells in recurrent spontaneous abortion (RSA). We aim to investigate the effect of SNHG1 on the phenotype of trophoblast cells during RSA. The RSA mice were established by mating female CBA/J mice with male DBA/2 mice. Microarray analysis was applied in RSA mice, and SNHG1 was identified as a significantly downregulated lncRNA. SNHG1 improved pregnancy outcome and reduced embryo resorption in RSA mice. Trophoblast cell proliferation, apoptosis, migration, and invasion were investigated by CCK8, EdU, TUNEL, wound healing, and Transwell assays. SNHG1 promoted proliferation, migration, and invasion of trophoblast cells, and reduced apoptosis. Mechanistically, SNHG1 bound to miR-183-5p in trophoblast cells. Moreover, miR-183-5p directly targeted ZEB2. Rescue experiment showed that ZEB2 silencing reversed the ameliorative effect of SNHG1 on pregnancy outcome and the promotion of trophoblast activity in RSA mice by impaired the Wnt/β-catenin pathway. In conclusion, we found that SNHG1 plays a critical role in the progression of RSA via miR-183-5p/ZEB2 and Wnt/β-catenin signaling. It has potential to be a therapeutic marker of RSA. 相似文献
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Eikan Mishima Daisuke Jinno Yasutoshi Akiyama Kunihiko Itoh Shinnosuke Nankumo Hisato Shima Koichi Kikuchi Yoichi Takeuchi Alaa Elkordy Takehiro Suzuki Kuniyasu Niizuma Sadayoshi Ito Yoshihisa Tomioka Takaaki Abe 《PloS one》2015,10(11)
The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism. 相似文献
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WeiShen Aik John S. Scotti Hwanho Choi Lingzhi Gong Marina Demetriades Christopher J. Schofield Michael A. McDonough 《Nucleic acids research》2014,42(7):4741-4754
ALKBH5 is a 2-oxoglutarate (2OG) and ferrous iron-dependent nucleic acid oxygenase (NAOX) that catalyzes the demethylation of N6-methyladenine in RNA. ALKBH5 is upregulated under hypoxia and plays a role in spermatogenesis. We describe a crystal structure of human ALKBH5 (residues 66–292) to 2.0 Å resolution. ALKBH566–292 has a double-stranded β-helix core fold as observed in other 2OG and iron-dependent oxygenase family members. The active site metal is octahedrally coordinated by an HXD…H motif (comprising residues His204, Asp206 and His266) and three water molecules. ALKBH5 shares a nucleotide recognition lid and conserved active site residues with other NAOXs. A large loop (βIV–V) in ALKBH5 occupies a similar region as the L1 loop of the fat mass and obesity-associated protein that is proposed to confer single-stranded RNA selectivity. Unexpectedly, a small molecule inhibitor, IOX3, was observed covalently attached to the side chain of Cys200 located outside of the active site. Modelling substrate into the active site based on other NAOX–nucleic acid complexes reveals conserved residues important for recognition and demethylation mechanisms. The structural insights will aid in the development of inhibitors selective for NAOXs, for use as functional probes and for therapeutic benefit. 相似文献
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ALKBH4 is a versatile demethylase capable of catalyzing the demethylation of monomethylated lysine-84 on actin and N6-methyladenine in DNA. In this study, we conducted a quantitative proteomic experiment to reveal the altered expression of proteins in HEK293T cells upon genetic ablation of ALKBH4. Our results showed markedly diminished levels of GSTP1 and HSPB1 proteins in ALKBH4-depleted cells, which emanate from an augmented expression level of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and the ensuing elevated cytosine methylation in the promoter regions of GSTP1 and HSPB1 genes. Together, our results revealed a role of ALKBH4 in modulating DNA cytosine methylation through regulating the expression level of DNMT1 protein. 相似文献
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Tina A. Müller Sarah L. Struble Katheryn Meek Robert P. Hausinger 《Biochemical and biophysical research communications》2018,495(1):98-103
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. 相似文献