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    Meclofenamic acid promotes cisplatin-induced acute kidney injury by inhibiting fat mass and obesity-associated protein-mediated mA abrogation in RNA. Zhou Peihui,Wu Ming,Ye Chaoyang,Xu Qingqing,Wang Li The Journal of biological chemistry The role of RNA methylation on the sixth N atom of adenylate (mA) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the mA modification in cisplatin-induced AKI. Here, we aimed to determine FTO's role in AKI. We induced AKI in c57BL/6 mice by intraperitoneal cisplatin injection and treated the animal with vehicle or an FTO inhibitor meclofenamic acid (MA) for 3 days. Moreover, as an model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases mA levels and MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased mA levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased mA in HK2 cells. FTO protein overexpression reduced mA levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in mA levels and apoptosis rates. In agreement, overexpression of the mA-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced mA levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both and , and FTO overexpression reduced p53 expression and reversed the MA-induced p53 increase in AKI. In conclusion, reduced renal FTO expression in cisplatin-induced AKI increases RNA mA levels and aggravates renal damages. 10.1074/jbc.RA119.011009
    Identification of Specific -Methyladenosine RNA Demethylase FTO Inhibitors by Single-Quantum-Dot-Based FRET Nanosensors. Zhang Yan,Li Qing-Nan,Zhou Kaiyue,Xu Qinfeng,Zhang Chun-Yang Analytical chemistry The fat mass and obesity-associated enzyme (FTO) can catalyze the demethylation of -methyladenosine (mA) residues in mRNA, regulates the cellular level of mA modification, and plays a critical role in human obesity and cancers. Herein, we develop a single-quantum-dot (QD)-based fluorescence resonance energy transfer (FRET) sensor for the identification of specific FTO demethylase inhibitors. The FTO-mediated demethylation of mA can induce the cleavage of demethylated DNA to generate the biotinylated DNA fragments, which may function as capture probes to assemble the Cy5-labeled reporter probes onto the QD surface, enabling the occurrence of FRET between the QD and Cy5. The presence of inhibitors can inhibit the FTO demethylation and consequently abolish FRET between the QD and Cy5. The inhibition effect of inhibitors upon FTO demethylation can be simply evaluated by monitoring the decrease of Cy5 counts. We use this nanosensor to screen several small-molecule inhibitors and identify diacerein as a highly selective inhibitor of FTO. Diacerein can inhibit the demethylation activity of endogenous FTO in HeLa cells. Interestingly, diacerein is neither a structural mimic of 2-oxoglutarate (2-OG) nor a chelator of metal ions, and it can selectively inhibit FTO demethylation by competitively binding the mA-containing substrate. 10.1021/acs.analchem.0c02828
    Changes in gene expression associated with FTO overexpression in mice. Merkestein Myrte,McTaggart James S,Lee Sheena,Kramer Holger B,McMurray Fiona,Lafond Mathilde,Boutens Lily,Cox Roger,Ashcroft Frances M PloS one Single nucleotide polymorphisms in the first intron of the fat-mass-and-obesity-related gene FTO are associated with increased body weight and adiposity. Increased expression of FTO is likely underlying this obesity phenotype, as mice with two additional copies of Fto (FTO-4 mice) exhibit increased adiposity and are hyperphagic. FTO is a demethylase of single stranded DNA and RNA, and one of its targets is the m6A modification in RNA, which might play a role in the regulation of gene expression. In this study, we aimed to examine the changes in gene expression that occur in FTO-4 mice in order to gain more insight into the underlying mechanisms by which FTO influences body weight and adiposity. Our results indicate an upregulation of anabolic pathways and a downregulation of catabolic pathways in FTO-4 mice. Interestingly, although genes involved in methylation were differentially regulated in skeletal muscle of FTO-4 mice, no effect of FTO overexpression on m6A methylation of total mRNA was detected. 10.1371/journal.pone.0097162
    N-methyladenosine mediates the cellular proliferation and apoptosis via microRNAs in arsenite-transformed cells. Gu Shiyan,Sun Donglei,Dai Huangmei,Zhang Zunzhen Toxicology letters N-methyladenosine (mA) modification is implicated to play an important role in cellular biological processes, but its regulatory mechanisms in arsenite-induced carcinogenesis are largely unknown. Here, human bronchial epithelial (HBE) cells were chronically treated with 2.5 μM arsenite sodium (NaAsO) for about 13 weeks and these cells were identified with malignant phenotype which was demonstrated by increased levels of cellular proliferation, percentages of plate colony formation and soft agar clone formation, and high potential of resistance to apoptotic induction. Our results firstly demonstrated that mA modification on RNA was significantly increased in arsenite-transformed cells and this modification may be synergistically regulated by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), Wilms tumor 1-associated protein (WTAP) and Fat mass and obesity-associated protein (FTO). In addition, knocking down of METTL3 in arsenite-transformed cells can dramatically reverse the malignant phenotype, which was manifested by lower percentages of clone and colony formation as well as higher rates of apoptotic induction. Given the critical roles of miRNAs in cellular proliferation and apoptosis, miRNAs regulated by mA in arsenite-transformed cells were analyzed by Venn diagram and KEGG pathway in this study. The results showed that these mA-mediated miRNAs can regulate pathways which are closely associated with cellular proliferation and apoptosis, implicating that these miRNAs may be the critical bridge by which mA mediates dysregulation of cell survival and apoptosis in arsenite-transformed cells. Taken together, our results firstly demonstrated the significant role of mA in the prevention of tumor occurrence and progression induced by arsenite. 10.1016/j.toxlet.2018.04.018
    FTO - A Common Genetic Basis for Obesity and Cancer. Lan Ning,Lu Ying,Zhang Yigan,Pu Shuangshuang,Xi Huaze,Nie Xin,Liu Jing,Yuan Wenzhen Frontiers in genetics In recent years, the prevalence of obesity and cancer have been rising. Since this poses a serious threat to human health, the relationship between the two has attracted much attention. This study examined whether fat mass and obesity-associated () genes are linked, taking into account a Genome-wide Association Study (GWAS) that revealed multiple single nucleotide polymorphism sites (SNPs) of the gene, indicating an association between obesity and cancer in different populations. FTO proteins have been proved to participate in adipogenesis and tumorigenesis with post-transcriptional regulation of downstream molecular expression or through the target of the mammalian target protein rapamycin (mTOR). FTO inhibitors have also been found to share anti-obesity and anti-cancer effects . In this review, we comprehensively discuss the correlation between obesity and cancer by measuring gene polymorphism, as well as the molecular mechanism involved in these diseases, emphasizing FTO as the common genetic basis of obesity and cancer. 10.3389/fgene.2020.559138
    Loss of m A on FAM134B promotes adipogenesis in porcine adipocytes through m A-YTHDF2-dependent way. Cai Min,Liu Qing,Jiang Qin,Wu Ruifan,Wang Xinxia,Wang Yizhen IUBMB life N -methyladenosine (m A) mRNA modification plays an important role in adipogenesis, but its role on single gene remains unexplored. Family with Sequence Similarity 134, Member B (FAM134B) is a cis-Golgi transmembrane protein that known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in lipid homeostasis and was identified as its significant m A level difference between Chinese local Jinhua pigs and Landrace through RNA-sequence. Here, we construct the non-m A FAM134B coding sequence (CDS) plasmid (FAM134B-MUT) and found one important m A site on its CDS. Expression of FAM134B-MUT was more effective in promoting porcine preadipocytes adipogenic differentiation and lipid deposition than wild-type FAM134B (FAM134B-WT) both in early and ultimate differentiation stage. FAM134B-MUT functions better in promoting fat deposition by upregulating peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein (C/EBPα) level. The m A reader protein YTH m A RNA binding protein 2 (YTHDF2) interacts with FAM134B mRNA and down regulated its protein level. These results demonstrate that FAM134B was the target of YTHDF2, which may recognize and binds the m A site of FAM134B to reduce its mRNA lifetime and reduce its protein abundance. © 2018 IUBMB Life, 71(5):580-586, 2019. 10.1002/iub.1974
    RUNX1T1 rs34269950 is associated with obesity and metabolic syndrome. Zhou Yuling,Hambly Brett D,Simmons David,McLachlan Craig S QJM : monthly journal of the Association of Physicians BACKGROUND:Runt-related transcription factor 1 (RUNX1T1) isoforms are involved in adipogenesis. RUNX1T1 is mediated by the fat mass and obesity-associated (FTO). However, the extent to which RUNX1T1 single nucleotide polymorphisms (SNPs), are associated with obesity risk or metabolic abnormalities in a community population basis is unknown. METHODS:Samples were obtained from the Australian Crossroads study bio-bank. SNPs located in the coding region and 3'untranslated regions of RUNX1T1 with minor allele frequency (MAF) ≥ 0.05 were analysed using Taqman genotyping assays. RESULTS:8 candidate SNPs were genotyped successfully in 1440 participants. Of these SNPs only rs34269950 located in the "RRACH" motif, the most common N6-methyladenosine (m6A) methylation modification site (recognized by FTO), was significantly associated with obesity risk and metabolic abnormalities. Specifically, compared to AA genotype, rs34269950 del/del genotype was associated with a 1.47 (95% CI: 1.01-2.14, P = 0.042) fold higher rate of obesity risk. Additionally, the del/del genotype was associated with a 60% increased risk of metabolic syndrome (MetS) (OR = 1.60, 95% CI: 1.10 - 2.32, P = 0.015), in comparison to the AA genotype. Finally, rs34269950 del/del increased the risk of a larger waist circumference (OR = 1.65, 95% CI: 1.15 - 2.36, P = 0.007), but not other components of MetS. CONCLUSION:Our study demonstrates that RUNX1T1 rs34269950, located in a potential FTO recognition motif, is significantly associated with waist circumference. This provides novel evidence to suggest SNPs located in RRACH motif may be involved in RNA m6A modification and mechanistic pathways that influence abdominal obesity. 10.1093/qjmed/hcaa208
    Kinase GSK3β functions as a suppressor in colorectal carcinoma through the FTO-mediated MZF1/c-Myc axis. Zhang Zeyan,Gao Qianfu,Wang Shanchao Journal of cellular and molecular medicine Colorectal carcinoma (CRC) poses heavy burden to human health and has an increasing incidence. Currently, the existing biomarkers for CRC bring about restrained clinical benefits. GSK3β is reported to be a novel therapeutic target for this disease but with undefined molecular mechanisms. Thus, we aimed to investigate the regulatory effect of GSK3β on CRC progression via FTO/MZF1/c-Myc axis. Firstly, the expression patterns of GSK3β, FTO, MZF1 and c-Myc were determined after sample collection. Lowly expressed GSK3β but highly expressed FTO, MZF1 and c-Myc were found in CRC. After transfection of different overexpressed and interference plasmids, the underlying mechanisms concerning GSK3β in CRC cell functions were analysed. Additionally, the effect of GSK3β on FTO protein stability was assessed followed by detection of MZF1 m6A modification and MZF1-FTO interaction. Mechanistically, GSK3β mediated ubiquitination of demethylase FTO to reduce FTO expression. Besides, GSK3β inhibited MZF1 expression by mediating FTO-regulated m6A modification of MZF1 and then decreased the proto-oncogene c-Myc expression, thus hampering CRC cell proliferation. We also carried out in vivo experiment to verify the regulatory effect of GSK3β on CRC via FTO-mediated MZF1/c-Myc axis. It was found that GSK3β inhibited CRC growth in vivo which was reversed by overexpressing c-Myc. Taken together, our findings indicate that GSK3β suppresses the progression of CRC through FTO-regulated MZF1/c-Myc axis, shedding light onto a new possible pathway by which GSK3β regulates CRC. 10.1111/jcmm.16291
    Dysregulation of USP18/FTO/PYCR1 signaling network promotes bladder cancer development and progression. Song Wei,Yang Ke,Luo Jianjun,Gao Zhiyong,Gao Yunliang Aging N6-methyladenosine refers to a methylation of adenosine base at the 6 nitrogen position, which is the dominant methylation modification in both message and non-coding RNAs. Dysregulation of RNA m6A methylation causes tumorigenesis in humans. The key N6-methyladenosine demethylase fat-mass and obesity-associated protein (FTO) is negatively correlated with the overall survival of bladder cancer patients, but the underlying mechanism remains poorly understood. In this study, we demonstrated that the post-translational deubiquitination by USP18 up-regulates the protein but not mRNA of FTO in bladder cancer tissues and cells. As a result, FTO decreased N6-methyladenosine methylation level in through its demethylase enzymatic activity and stabilized transcript to promote bladder cancer initiation and progression. Our work shows the importance of N6-methyladenosine RNA modification in bladder cancer development, and highlights UPS18/FTO/PYCR1 signaling network as potential therapeutic targets of bladder cancer. 10.18632/aging.202359
    The FTO/miR-181b-3p/ARL5B signaling pathway regulates cell migration and invasion in breast cancer. Xu Yuanyuan,Ye Shuang,Zhang Nan,Zheng Shuhui,Liu Huatao,Zhou Kewen,Wang Ling,Cao Yue,Sun Peng,Wang Tinghuai Cancer communications (London, England) BACKGROUND:N6-methyladenosine (m A) RNA modification has been demonstrated to be a significant regulatory process in the progression of various tumors, including breast cancer. Fat mass and obesity-associated (FTO) enzyme, initially known as the obesity-related protein, is the first identified m A demethylase. However, the relationship between FTO and breast cancer remains controversial. In this study, we aimed to elucidate the role and clinical significance of FTO in breast cancer and to explore the underlying mechanism. METHODS:We first investigated the expression of FTO in breast cancer cell lines and tissues by quantitative reverse transcription-PCR (qRT-PCR), Western blotting, and immunohistochemistry. Wound healing assay and Transwell assay were performed to determine the migration and invasion abilities of SKBR3 and MDA-MB453 cells with either knockdown or overexpression of FTO. RNA sequencing (RNA-seq) was conducted to decipher the downstream targets of FTO. qRT-PCR, luciferase reporter assay, and Western blotting were employed to confirm the existence of the FTO/miR-181b-3p/ARL5B axis. The biological function of ADP ribosylation factor like GTPase 5B (ARL5B) in breast cancer cells was evaluated by wound healing assay and Transwell invasion assay. RESULTS:High FTO expression was observed in human epidermal growth factor receptor 2 (HER2)-positive breast cancer, predicting advanced progression (tumor size [P < 0.001], nuclear grade [P = 0.001], peritumoral lymphovascular invasion [P < 0.001), lymph node metastasis [P = 0.002], and TNM stage [P = 0.001]) and poor prognosis. Moreover, FTO promoted cell invasion and migration in vitro. Mechanistically, RNA-seq and further confirmation studies suggested that FTO up-regulated ARL5B by inhibiting miR-181b-3p. We further verified that ARL5B also displayed carcinogenic activity in breast cancer cells. CONCLUSION:Our work demonstrated the carcinogenic activity of FTO in promoting the invasion and migration of breast cancer cells via the FTO/miR-181b-3p/ARL5B signaling pathway. 10.1002/cac2.12075
    N6-methyladenosine demethylase FTO suppresses clear cell renal cell carcinoma through a novel FTO-PGC-1α signalling axis. Zhuang Changshui,Zhuang Chengle,Luo Xiaomin,Huang Xinbo,Yao Lv,Li Jianfa,Li Yawen,Xiong Tiefu,Ye Jing,Zhang Fangting,Gui Yaoting Journal of cellular and molecular medicine The abundant and reversible N6-methyladenosine (m6A) RNA modification and its modulators have important roles in regulating various gene expression and biological processes. Here, we demonstrate that fat mass and obesity associated (FTO), as an m6A demethylase, plays a critical anti-tumorigenic role in clear cell renal cell carcinoma (ccRCC). FTO is suppressed in ccRCC tissue. The low expression of FTO in human ccRCC correlates with increased tumour severity and poor patient survival. The Von Hippel-Lindau-deficient cells expressing FTO restores mitochondrial activity, induces oxidative stress and ROS production and shows impaired tumour growth, through increasing expression of PGC-1α by reducing m6A levels in its mRNA transcripts. Our work demonstrates the functional importance of the m6A methylation and its modulator, and uncovers a critical FTO-PGC-1α axis for developing effective therapeutic strategies in the treatment of ccRCC. 10.1111/jcmm.14128
    The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation. Zhang Meizi,Zhang Ying,Ma Jun,Guo Feima,Cao Qian,Zhang Yu,Zhou Bin,Chai Jijie,Zhao Wenqing,Zhao Renbin PloS one FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N6-methyladenosine (m6A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m6A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ. 10.1371/journal.pone.0133788
    Downregulation of Fat Mass and Obesity Associated (FTO) Promotes the Progression of Intrahepatic Cholangiocarcinoma. Rong Zhuo-Xian,Li Zhi,He Jun-Ju,Liu Li-Yu,Ren Xin-Xin,Gao Jie,Mu Yun,Guan Yi-Di,Duan Yu-Mei,Zhang Xiu-Ping,Zhang De-Xiang,Li Nan,Deng Yue-Zhen,Sun Lun-Quan Frontiers in oncology Intrahepatic cholangiocarcinoma (ICC) ranks as the second most malignant type of primary liver cancer with a high degree of incidence and a very poor prognosis. Fat mass and obesity-associated protein (FTO) functions as an eraser of the RNA mA modification, but its roles in ICC tumorigenesis and development remain unknown. We showed here that the protein level of FTO was downregulated in clinical ICC samples and cell lines and that FTO expression was inversely correlated with the expression of CA19-9 and micro-vessel density (MVD). A Kaplan-Meier survival analysis showed that a low expression of predicted poor prognosis in ICC. , decreased endogenous expression of obviously reduced apoptosis of ICC cells. Moreover, suppressed the anchorage-independent growth and mobility of ICC cells. Through mining the database, FTO was found to regulate the integrin signaling pathway, inflammation signaling pathway, epidermal growth factor receptor (EGFR) signaling pathway, angiogenesis, and the pyrimidine metabolism pathway. RNA decay assay showed that oncogene mRNA stability was impaired by FTO. In addition, the overexpression of FTO suppressed tumor growth . In conclusion, our study demonstrated the critical roles of FTO in ICC. 10.3389/fonc.2019.00369
    Fat mass and obesity-associated (FTO) protein regulates adult neurogenesis. Li Liping,Zang Liqun,Zhang Feiran,Chen Junchen,Shen Hui,Shu Liqi,Liang Feng,Feng Chunyue,Chen Deng,Tao Huikang,Xu Tianlei,Li Ziyi,Kang Yunhee,Wu Hao,Tang Lichun,Zhang Pumin,Jin Peng,Shu Qiang,Li Xuekun Human molecular genetics Fat mass and obesity-associated gene (FTO) is a member of the Fe (II)- and oxoglutarate-dependent AlkB dioxygenase family and is linked to both obesity and intellectual disability. The role of FTO in neurodevelopment and neurogenesis, however, remains largely unknown. Here we show that FTO is expressed in adult neural stem cells and neurons and displays dynamic expression during postnatal neurodevelopment. The loss of FTO leads to decreased brain size and body weight. We find that FTO deficiency could reduce the proliferation and neuronal differentiation of adult neural stem cells in vivo, which leads to impaired learning and memory. Given the role of FTO as a demethylase of N6-methyladenosine (m6A), we went on to perform genome-wide m6A profiling and observed dynamic m6A modification during postnatal neurodevelopment. The loss of FTO led to the altered expression of several key components of the brain derived neurotrophic factor pathway that were marked by m6A. These results together suggest FTO plays important roles in neurogenesis, as well as in learning and memory. 10.1093/hmg/ddx128
    Increased N6-methyladenosine causes infertility is associated with FTO expression. Ding Chenyue,Zou Qinyan,Ding Jie,Ling Mingfa,Wang Wei,Li Hong,Huang Boxian Journal of cellular physiology The N6-methyladenosine (m6A) modification plays a central role in epigenetic regulation of the mammalian transcriptome. m6A can be demethylated by the fat mass- and obesity-associated (FTO) protein and the α-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) protein. Much less is known about that whether m6A content is involved in POI (premature ovarian insufficiency) disease. In this case-controlled study, 69 POI and 53 tubal occlusion patients were recruited from the reproduction centers in our hospital. For the POI animal model experiment, ovarian tissue was obtained from ten POI and nine healthy mice. An m6A test kit was developed to determine the m6A content in the RNA, and qPCR and western blot were used to examine the mRNA and protein expression levels of FTO and ALKBH5. FACS was used to measure the levels of proliferation and apoptosis, and siRNA was used to establish FTO and ALKBH5 knockdown cell lines. Our results showed that the m6A content in the RNA from POI patients and POI mice was significantly higher than control groups and that POI was characterized by the content of m6A. The mRNA and protein expression levels of FTO were significantly lower in the POI patients than control group and were associated with a risk of POI. These data suggest that the decreased mRNA and protein expression levels of FTO may be responsible for the increase in m6A in POI, which may further increase the risk of complications of POI. High m6A should be investigated further as a novel potential biomarker of POI. 10.1002/jcp.26507
    Regulation of N6-Methyladenosine in the Differentiation of Cancer Stem Cells and Their Fate. Xu Ya,Liu Jing,Chen Wen-Jia,Ye Qian-Qian,Chen Wen-Tian,Li Chun-Lan,Wu Hua-Tao Frontiers in cell and developmental biology N6-methyladenosine (m6A) is one of the most common internal RNA modifications in eukaryotes. It is a dynamic and reversible process that requires an orchestrated participation of methyltransferase, demethylase, and methylated binding protein. m6A modification can affect RNA degradation, translation, and microRNA processing. m6A plays an important role in the regulation of various processes in living organisms. In addition to being involved in normal physiological processes such as sperm development, immunity, fat differentiation, cell development, and differentiation, it is also involved in tumor progression and stem cell differentiation. Curiously enough, cancer stem cells, a rare group of cells present in malignant tumors, retain the characteristics of stem cells and play an important role in the survival, proliferation, metastasis, and recurrence of cancers. Recently, studies demonstrated that m6A participates in the self-renewal and pluripotent regulation of these stem cells. However, considering that multiple targets of m6A are involved in different physiological processes, the exact role of m6A in cancer progression remains controversial. This article focuses on the mechanism of m6A and its effects on the differentiation of cancer stem cells, to provide a basis for elucidating the tumorigenesis mechanisms and exploring new potential therapeutic approaches. 10.3389/fcell.2020.561703
    FTO, m A , and the hypothesis of reversible epitranscriptomic mRNA modifications. Mauer Jan,Jaffrey Samie R FEBS letters The fate of mRNA is regulated by epitranscriptomic nucleotide modifications, the most abundant of which is N -methyladenosine (m A). Although the pattern and distribution of m A in mRNA is mediated by specific methyltransferases, a recent hypothesis is that specific demethylases or 'erasers' allow m A to be dynamically reversed by signaling pathways. In this Review, we discuss the data in support and against this model. New insights into the function of fat mass and obesity-associated protein (FTO), the original enzyme thought to be an m A eraser, reveal that its physiologic target is not m A, but instead is N ,2'-O-dimethyladenosine (m A ). Another m A demethylase, ALKBH5, appears to have functions limited to sperm development in normal mice. Overall, the majority of the data suggest that m A is generally not reversible, although m A may be susceptible to demethylation in pathophysiological states such as cancer. 10.1002/1873-3468.13092
    Preparation of Human Nuclear RNA m⁶A Methyltransferases and Demethylases and Biochemical Characterization of Their Catalytic Activity. Liu Jianzhao,Yue Yanan,He Chuan Methods in enzymology N(6)-Methyladenosine (m(6)A) represents the most prevalent internal modification in messenger and long noncoding RNAs. There has been a surge of interest toward understanding the biological significance of m(6)A modification. In this chapter, we describe the methods for biochemically studying the recently uncovered m(6)A methyltransferases (METTL3 and METTL14) and demethylases (FTO and ALKBH5). How to express these proteins, perform their biochemistry reactions against various RNA probes, and characterize the methylation and demethylation activity will be discussed. 10.1016/bs.mie.2015.03.013
    Identification of Flavin Mononucleotide as a Cell-Active Artificial N -Methyladenosine RNA Demethylase. Xie Li-Jun,Yang Xiao-Ti,Wang Rui-Li,Cheng Hou-Ping,Li Zhi-Yan,Liu Li,Mao Lanqun,Wang Ming,Cheng Liang Angewandte Chemie (International ed. in English) N -Methyladenosine (m A) represents a common and highly dynamic modification in eukaryotic RNA that affects various cellular pathways. Natural dioxygenases such as FTO and ALKBH5 are enzymes that demethylate m A residues in mRNA. Herein, the first identification of a small-molecule modulator that functions as an artificial m A demethylase is reported. Flavin mononucleotide (FMN), the metabolite produced by riboflavin kinase, mediates substantial photochemical demethylation of m A residues of RNA in live cells. This study provides a new perspective to the understanding of demethylation of m A residues in mRNA and sheds light on the development of powerful small molecules as RNA demethylases and new probes for use in RNA biology. 10.1002/anie.201900901
    Meclofenamic acid selectively inhibits FTO demethylation of m6A over ALKBH5. Huang Yue,Yan Jingli,Li Qi,Li Jiafei,Gong Shouzhe,Zhou Hu,Gan Jianhua,Jiang Hualiang,Jia Gui-Fang,Luo Cheng,Yang Cai-Guang Nucleic acids research Two human demethylases, the fat mass and obesity-associated (FTO) enzyme and ALKBH5, oxidatively demethylate abundant N(6)-methyladenosine (m(6)A) 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 m(6)A-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 m(6)A 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. 10.1093/nar/gku1276
    FTO reduces mitochondria and promotes hepatic fat accumulation through RNA demethylation. Kang Huifang,Zhang Zhiwang,Yu Lin,Li Yixing,Liang Mingzhen,Zhou Lei Journal of cellular biochemistry Fat mass and obesity-associated protein (FTO) is a RNA demethylase, whether FTO regulates fat metabolism through its demethylation is unclear. The results of this study confirmed that N6-methyladenosine (m A) is associated with fat accumulation both in vivo and in vitro. The data showed that FTO down-regulated m A levels, decreased mitochondrial content, and increased triglyceride (TG) deposition. However, an FTO (R316A) mutant lacking demethylation activity could not regulate mitochondria and TG content, indicating that FTO affects mitochondrial content and fat metabolism by modulating m A levels in hepatocytes. In addition, the regulatory roles of cycloleucine (methylation inhibitor) and betaine (methyl donor) could regulate m A levels and fat deposition. This work clarified that the demethylation function of FTO plays an essential role in the fat metabolism of hepatocytes and links the epigenetic modification of RNA with fat deposition, thereby providing a new target (m A) for regulation of hepatic fat metabolism. 10.1002/jcb.26746
    The N6-methyladenosine RNA modification in acute myeloid leukemia. Yankova Eliza,Aspris Demetrios,Tzelepis Konstantinos Current opinion in hematology PURPOSE OF REVIEW:In recent years, the N6-methyladenosine (m6A) modification of RNA has been shown to play an important role in the development of acute myeloid leukemia (AML) and the maintenance of leukemic stem cells (LSCs). In this review we summarise the recent findings in the field of epitranscriptomics related to m6A and its relevance in AML. RECENT FINDINGS:Recent studies have focused on the role of m6A regulators in the development of AML and their potential as translational targets. The writer Methyltransferase Like 3 and its binding partner Methyltransferase Like 14, as well as the reader YTH domain-containing family protein 2, were shown to be vital for LSC survival, and their loss has detrimental effects on AML cells. Similar observations were made with the demethylases fat mass and obesity-associated protein and AlkB homologue 5 RNA demethylase. Of importance, loss of any of these genes has little to no effect on normal hemopoietic stem cells, suggesting therapeutic potential. SUMMARY:The field of epitranscriptomics is still in its infancy and the importance of m6A and other RNA-modifications in AML will only come into sharper focus. The development of therapeutics targeting RNA-modifying enzymes may open up new avenues for treatment of such malignancies. 10.1097/MOH.0000000000000636
    RNA mA methylation regulates the ultraviolet-induced DNA damage response. Xiang Yang,Laurent Benoit,Hsu Chih-Hung,Nachtergaele Sigrid,Lu Zhike,Sheng Wanqiang,Xu Chuanyun,Chen Hao,Ouyang Jian,Wang Siqing,Ling Dominic,Hsu Pang-Hung,Zou Lee,Jambhekar Ashwini,He Chuan,Shi Yang Nature Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (mA) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A) transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3) and the demethylase FTO (fat mass and obesity-associated protein). In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of mA in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA mA modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which mA RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival. 10.1038/nature21671
    Epigallocatechin gallate targets FTO and inhibits adipogenesis in an mRNA mA-YTHDF2-dependent manner. Wu Ruifan,Yao Yongxi,Jiang Qin,Cai Min,Liu Qing,Wang Yizhen,Wang Xinxia International journal of obesity (2005) BACKGROUND/OBJECTIVE:N-methyladenosine (mA) modification of mRNA plays a role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, plays a critical role in anti-obesity and anti-adipogenesis. METHODS:High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) was performed to determine the mA levels in 3T3-L1 preadipocytes. The effects of EGCG on the mA levels in specific genes were determined by methylated RNA immunoprecipitation coupled with quantitative real-time PCR (meRIP-qPCR). Several adipogenesis makers and cell cycle genes were analyzed by quantitative real-time PCR (qPCR) and western blotting. Lipid accumulation was evaluated by oil red O staining. All measurements were performed at least for three times. RESULTS:Here we showed that EGCG inhibited adipogenesis by blocking the mitotic clonal expansion (MCE) at the early stage of adipocyte differentiation. Exposing 3T3-L1 cells to EGCG reduced the expression of fat mass and obesity-associated (FTO) protein, an mA demethylase, which led to increased overall levels of RNA mA methylation. Cyclin A2 (CCNA2) and cyclin dependent kinase 2 (CDK2) play vital roles in MCE. The mA levels of CCNA2 and CDK2 mRNA were dramatically enhanced by EGCG. Interestingly, EGCG increased the expression of YTH N-methyladenosine RNA binding protein 2 (YTHDF2), which recognized and decayed methylated mRNAs, resulting in decreased protein levels of CCNA2 and CDK2. As a result, MCE was blocked and adipogenesis was inhibited. FTO overexpression and YTHDF2 knockdown in 3T3-L1 cells significantly increased CCNA2 and CDK2 protein levels and ameliorated the EGCG-induced adipogenesis inhibition. Thus, mA-dependent CCNA2 and CDK2 expressions mediated by FTO and YTHDF2 contributed to EGCG-induced adipogenesis inhibition. CONCLUSION:Our findings provide mechanistic insights into how mA is involved in the EGCG regulation of adipogenesis and shed light on its anti-obesity effect. 10.1038/s41366-018-0082-5
    R-2HG Exhibits Anti-tumor Activity by Targeting FTO/mA/MYC/CEBPA Signaling. Su Rui,Dong Lei,Li Chenying,Nachtergaele Sigrid,Wunderlich Mark,Qing Ying,Deng Xiaolan,Wang Yungui,Weng Xiaocheng,Hu Chao,Yu Mengxia,Skibbe Jennifer,Dai Qing,Zou Dongling,Wu Tong,Yu Kangkang,Weng Hengyou,Huang Huilin,Ferchen Kyle,Qin Xi,Zhang Bin,Qi Jun,Sasaki Atsuo T,Plas David R,Bradner James E,Wei Minjie,Marcucci Guido,Jiang Xi,Mulloy James C,Jin Jie,He Chuan,Chen Jianjun Cell R-2-hydroxyglutarate (R-2HG), produced at high levels by mutant isocitrate dehydrogenase 1/2 (IDH1/2) enzymes, was reported as an oncometabolite. We show here that R-2HG also exerts a broad anti-leukemic activity in vitro and in vivo by inhibiting leukemia cell proliferation/viability and by promoting cell-cycle arrest and apoptosis. Mechanistically, R-2HG inhibits fat mass and obesity-associated protein (FTO) activity, thereby increasing global N-methyladenosine (mA) RNA modification in R-2HG-sensitive leukemia cells, which in turn decreases the stability of MYC/CEBPA transcripts, leading to the suppression of relevant pathways. Ectopically expressed mutant IDH1 and S-2HG recapitulate the effects of R-2HG. High levels of FTO sensitize leukemic cells to R-2HG, whereas hyperactivation of MYC signaling confers resistance that can be reversed by the inhibition of MYC signaling. R-2HG also displays anti-tumor activity in glioma. Collectively, while R-2HG accumulated in IDH1/2 mutant cancers contributes to cancer initiation, our work demonstrates anti-tumor effects of 2HG in inhibiting proliferation/survival of FTO-high cancer cells via targeting FTO/mA/MYC/CEBPA signaling. 10.1016/j.cell.2017.11.031
    N-methyladenosine (mA) methylation in ischemia-reperfusion injury. Yao Weifeng,Han Xue,Ge Mian,Chen Chaojin,Xiao Xue,Li Haobo,Hei Ziqing Cell death & disease Ischemia-reperfusion (I/R) injury is common during surgery and often results in organ dysfunction. The mechanisms of I/R injury are complex, diverse, and not well understood. RNA methylation is a novel epigenetic modification that is involved in the regulation of various biological processes, such as immunity, response to DNA damage, tumorigenesis, metastasis, stem cell renewal, fat differentiation, circadian rhythms, cell development and differentiation, and cell division. Research on RNA modifications, specifically N6-methyladenosine (mA), have confirmed that they are involved in the regulation of organ I/R injury. In this review, we summarized current understanding of the regulatory roles and significance of mA RNA methylation in I/R injury in different organs. 10.1038/s41419-020-2686-7
    Fat mass and obesity-associated protein promotes the tumorigenesis and development of liver cancer. Ye Ziqi,Wang Shibing,Chen Wanyuan,Zhang Xin,Chen Jie,Jiang Jinying,Wang Mingshan,Zhang Li,Xuan Zixue Oncology letters Liver cancer is the fourth leading cause of cancer-associated mortality worldwide. Statistics indicate that the incidence of liver cancer has been increasing and that its prognosis remains poor. Fat mass and obesity-associated protein (FTO) is a demethylase that is involved in N6-methyladenosine (m6a) RNA modification; however, to the best of our knowledge, its role in tumorigenesis and development of liver cancer remains unknown. In the present study, cell proliferation, colony formation, apoptosis, Transwell and wound healing assays of small interfering (si)RNA-FTO HepG2 cells were performed, and the levels of m6A RNA methylation were assessed. Additionally, the prognostic value of FTO in liver cancer was analyzed using immunohistochemistry analysis. The results from the EpiQuik m6A RNA methylation quantitative assay revealed that knockdown of FTO increased the total m6A methylation level. Notably, FTO promoted the proliferation and migration of liver cancer cells. Additionally, FTO expression was upregulated in patients with liver cancer and was associated with a high Edmondson Grade, which served as an independent prognostic factor for liver cancer. Results from the Kaplan-Meier survival analysis revealed that low expression levels of FTO predicted a good prognosis. The 5-year overall survival of the low FTO expression group was 68% compared with 48% in the high FTO expression group (P=0.077). In conclusion, the present study suggested that FTO regulates the tumorigenesis and development of liver cancer. 10.3892/ol.2020.11673
    mRNA mA plays opposite role in regulating UCP2 and PNPLA2 protein expression in adipocytes. Wang Xinxia,Sun Baofa,Jiang Qin,Wu Ruifan,Cai Min,Yao Yongxi,Liu Qing,Shi Hailing,Feng Jie,Wang Yizhen International journal of obesity (2005) BACKGROUND/OBJECTIVE:N-methyladenosine (mA) modification of mRNA plays an important role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. SUBJECTS/METHODS:Using Jinhua and Landrace pigs as fat and lean models, we presented a comprehensive transcriptome-wide mA profiling in adipose tissues from these two pig breeds. Two differentially methylated genes were selected to explore the mechanisms of mA-mediated regulation of gene function. RESULTS:The ratio of mA/A in the layer of backfat (LB) was significantly higher in Landrace than that in Jinhua. Transcriptome-wide mA profiling revealed that mA modification on mRNA occurs in the conserved sequence motif of RRACH and that the pig transcriptome contains 0.53-0.91 peak per actively expressed transcript. The relative density of mA peaks in the 3'UTR were higher than in 5'UTR. Genes with common mA peaks from both Landrace (L-LB) and Jinhua (J-LB) were enriched in RNA splicing and cellular lipid metabolic process. The unique mA peak genes (UMGs) from L-LB were mainly enriched in the extracellular matrix (ECM) and collagen catabolic process, whereas the UMGs from J-LB are mainly involved in RNA splicing, etc. Lipid metabolism processes were not significantly enriched in the UMGs from L-LB or J-LB. Uncoupling protein-2 (UCP2) and patatin-like phospholipase domain containing 2 (PNPLA2) were two of the UMGs in L-LB. Synonymous mutations (MUT) were conducted to reduce mA level of UCP2 and PNPLA2 mRNAs. Adipogenesis test showed that UCP2-MUT further inhibited adipogenesis, while PNPLA2-MUT promoted lipid accumulation compared with UCP2-WT and PNPLA2-WT, respectively. Further study showed mA negatively mediates UCP2 protein expression and positively mediates PNPLA2 protein expression. mA modification affects the translation of PNPLA2 most likely through YTHDF1, whereas UCP2 is likely neither the target of YTHDF2 nor the target of YTHDF1. CONCLUSION:Our data demonstrated a conserved and yet dynamically regulated mA methylome in pig transcriptomes and provided an important resource for studying the function of mA epitranscriptomic modification in obesity development. 10.1038/s41366-018-0027-z
    mA-RNA Demethylase FTO Inhibitors Impair Self-Renewal in Glioblastoma Stem Cells. Huff Sarah,Tiwari Shashi Kant,Gonzalez Gwendolyn M,Wang Yinsheng,Rana Tariq M ACS chemical biology -methyladenosine (mA) has emerged as the most abundant mRNA modification that regulates gene expression in many physiological processes. mA modification in RNA controls cellular proliferation and pluripotency and has been implicated in the progression of multiple disease states, including cancer. RNA mA methylation is controlled by a multiprotein "writer" complex including the enzymatic factor methyltransferase-like protein 3 (METTL3) that regulates methylation and two "eraser" proteins, RNA demethylase ALKBH5 (ALKBH5) and fat mass- and obesity-associated protein (FTO), that demethylate mA in transcripts. FTO can also demethylate ,2'--dimethyladenosine (mA), which is found adjacent to the mG cap structure in mRNA. FTO has recently gained interest as a potential cancer target, and small molecule FTO inhibitors such as meclofenamic acid have been shown to prevent tumor progression in both acute myeloid leukemia and glioblastoma models. However, current FTO inhibitors are unsuitable for clinical applications due to either poor target selectivity or poor pharmacokinetics. In this work, we describe the structure-based design, synthesis, and biochemical evaluation of a new class of FTO inhibitors. Rational design of 20 small molecules with low micromolar IC's and specificity toward FTO over ALKBH5 identified two competitive inhibitors FTO-02 and FTO-04. Importantly, FTO-04 prevented neurosphere formation in patient-derived glioblastoma stem cells (GSCs) without inhibiting the growth of healthy neural stem cell-derived neurospheres. Finally, FTO-04 increased mA and mA levels in GSCs consistent with FTO inhibition. These results support FTO-04 as a potential new lead for treatment of glioblastoma. 10.1021/acschembio.0c00841
    The M6A methyltransferase METTL3 promotes the development and progression of prostate carcinoma via mediating MYC methylation. Yuan Yan,Du Yang,Wang Lei,Liu Xiuheng Journal of Cancer N6-methyladenosine (mA) is the richest modification in mammalian messenger RNAs (mRNAs), and exerts key roles in many biological processes, including cancer development, whereas its roles in prostate carcinoma (PCa) remain to be unclear. Here, we found that mA modifications are increased in PCa and methyltransferase-like 3 (METTL3), but not other major mA modification genes including METTL14, fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5), was the major dysregulated gene associated with abnormal mA modification. In addition, METTL3 up-regulation acted as a poor prognostic factor for overall survival and disease-free survival in PCa patients. Knockdown of METTL3 significantly inhibited PCa cells proliferation, migration, and invasion. In addition, over-expression of METTL3, but not its catalytic mutant form, significantly promoted PCa cells growth and progression. Mechanistically, we revealed that METTL3 enhanced MYC(c-myc) expression by increasing mA levels of MYC mRNA transcript, leading to oncogenic functions in PCa. Importantly, PCa cells growth and progression inhibition by METTL3 knockdown were restored through over-expression of MYC. Our results uncovered a METTL3/mA/MYC axis and provided insight into the mechanisms of PCa progression. 10.7150/jca.42338
    mA Regulates Liver Metabolic Disorders and Hepatogenous Diabetes. Li Yuhuan,Zhang Qingyang,Cui Guanshen,Zhao Fang,Tian Xin,Sun Bao-Fa,Yang Ying,Li Wei Genomics, proteomics & bioinformatics N-methyladenosine (mA) is one of the most abundant modifications on mRNAs and plays important roles in various biological processes. The formation of mA is catalyzed by a methyltransferase complex (MTC) containing a key factor methyltransferase-like 3 (Mettl3). However, the functions of Mettl3 and mA modification in hepatic lipid and glucose metabolism remain unclear. Here, we showed that both Mettl3 expression and mA level increased in the livers of mice with high fat diet (HFD)-induced metabolic disorders. Overexpression of Mettl3 aggravated HFD-induced liver metabolic disorders and insulin resistance. In contrast, hepatocyte-specific knockout of Mettl3 significantly alleviated HFD-induced metabolic disorders by slowing weight gain, reducing lipid accumulation, and improving insulin sensitivity. Mechanistically, Mettl3 depletion-mediated mA loss caused extended RNA half-lives of metabolism-related genes, which consequently protected mice against HFD-induced metabolic syndrome. Our findings reveal a critical role of Mettl3-mediated mA in HFD-induced metabolic disorders and hepatogenous diabetes. 10.1016/j.gpb.2020.06.003
    R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/mA/PFKP/LDHB axis. Qing Ying,Dong Lei,Gao Lei,Li Chenying,Li Yangchan,Han Li,Prince Emily,Tan Brandon,Deng Xiaolan,Wetzel Collin,Shen Chao,Gao Min,Chen Zhenhua,Li Wei,Zhang Bin,Braas Daniel,Ten Hoeve Johanna,Sanchez Gerardo Javier,Chen Huiying,Chan Lai N,Chen Chun-Wei,Ann David,Jiang Lei,Müschen Markus,Marcucci Guido,Plas David R,Li Zejuan,Su Rui,Chen Jianjun Molecular cell R-2-hydroxyglutarate (R-2HG), a metabolite produced by mutant isocitrate dehydrogenases (IDHs), was recently reported to exhibit anti-tumor activity. However, its effect on cancer metabolism remains largely elusive. Here we show that R-2HG effectively attenuates aerobic glycolysis, a hallmark of cancer metabolism, in (R-2HG-sensitive) leukemia cells. Mechanistically, R-2HG abrogates fat-mass- and obesity-associated protein (FTO)/N-methyladenosine (mA)/YTH N-methyladenosine RNA binding protein 2 (YTHDF2)-mediated post-transcriptional upregulation of phosphofructokinase platelet (PFKP) and lactate dehydrogenase B (LDHB) (two critical glycolytic genes) expression and thereby suppresses aerobic glycolysis. Knockdown of FTO, PFKP, or LDHB recapitulates R-2HG-induced glycolytic inhibition in (R-2HG-sensitive) leukemia cells, but not in normal CD34 hematopoietic stem/progenitor cells, and inhibits leukemogenesis in vivo; conversely, their overexpression reverses R-2HG-induced effects. R-2HG also suppresses glycolysis and downregulates FTO/PFKP/LDHB expression in human primary IDH-wild-type acute myeloid leukemia (AML) cells, demonstrating the clinical relevance. Collectively, our study reveals previously unrecognized effects of R-2HG and RNA modification on aerobic glycolysis in leukemia, highlighting the therapeutic potential of targeting cancer epitranscriptomics and metabolism. 10.1016/j.molcel.2020.12.026
    The FTO mA demethylase inhibits the invasion and migration of prostate cancer cells by regulating total mA levels. Zhu Kai,Li Ying,Xu Yikai Life sciences AIMS:N6-Methyladenosine (mA) is the most frequent posttranscriptional modification and plays important roles in tumorigenesis and metastasis. The roles of fat mass and obesity-associated (FTO) in metabolic diseases have been widely explored. However, the molecular mechanisms and physiological functions of FTO in prostate cancer remain largely unknown. This study aimed to explore the exact functions of FTO in the progression of prostate cancer metastasis. MAIN METHODS:Dot blot and mA RNA methylation quantification assays were performed to determine mA levels. The protein and mRNA expression levels were detected using immunoblot (IB) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Cell invasion and migration abilities were measured using transwell and wound healing assays. Bioinformatics was used to measure the expression level of FTO and possible correlation between FTO levels and advanced tumor stage. Immunofluorescence (IF) was performed to measure the cellular localization of FTO. KEY FINDINGS:FTO was downregulated in prostate cancer tissues and cell lines, and the mA content was increased. Importantly, patients with lower FTO expression had advanced tumor stage and higher Gleason scores. Gain- and loss-of-function assays revealed that FTO inhibits prostate cancer cell invasion and migration in vitro. Moreover, we confirmed that FTO can decrease the total mA level. SIGNIFICANCE:The present study revealed that the FTO mA demethylase inhibits prostate cancer cell invasion and migration by regulating total mA levels. 10.1016/j.lfs.2021.119180
    TNF-α suppresses sweat gland differentiation of MSCs by reducing FTO-mediated mA-demethylation of Nanog mRNA. Wang Yihui,Wang Rui,Yao Bin,Hu Tian,Li Zhao,Liu Yufan,Cui Xiaoli,Cheng Liuhanghang,Song Wei,Huang Sha,Fu Xiaobing Science China. Life sciences An effect of inhibition of tumor necrosis factor-α (TNF-α) on differentiation of mesenchymal stromal cells (MSCs) has been demonstrated, but the exact mechanisms that govern MSCs differentiation remain to be further elucidated. Here, we show that TNF-α inhibits the differentiation of MSCs to sweat glands in a specific sweat gland-inducing environment, accompanied with reduced expression of Nanog, a core pluripotency factor. We elucidated that fat mass and obesity-associated protein (FTO)-mediated mA demethylation is involved in the regulation of MSCs differentiation potential. Exposure of MSCs to TNF-α reduced expression of FTO, which demethylated Nanog mRNA. Reduced expression of FTO increased Nanog mRNA methylation, decreased Nanog mRNA and protein expression, and significantly inhibited MSCs capacity for differentiation to sweat gland cells. Our finding is the first to elucidate the functional importance of mA modification in MSCs, providing new insights that the microenvironment can regulate the multipotency of MSCs at the post-transcriptional level. Moreover, to maintain differentiation capacity of MSCs by regulating mA modification suggested a novel potential therapeutic target for stem cell-mediated regenerative medicine. 10.1007/s11427-019-9826-7
    METTL3 inhibits hepatic insulin sensitivity via N6-methyladenosine modification of Fasn mRNA and promoting fatty acid metabolism. Xie Wei,Ma Lei Lei,Xu Yue Qing,Wang Bao Hua,Li Sai Mei Biochemical and biophysical research communications Type 2 diabetes (T2D) is characterized by lack of insulin, insulin resistance and high blood sugar. However, the underlying mechanisms of insulin resistance during T2D development remains unclear. As the most common mRNAs modification, N6-Methyladenosine (m6A) is involved in many of pathological processes in aging disease. However, it remains unclear whether m6A is involved in T2D development and what is the regulatory mechanism. This study is aimed to illustrate the roles of m6A and its methyltransferase METTL3 in the regulation of blood glucose homeostasis and insulin sensitivity. The results showed that m6A methylated RNA level and its N6-methyladenosine methylase METTL3 were consistently up-regulated in the liver tissues from patients with T2D. Moreover, both m6A methylated RNA and METTL3 levels showed positive correlation with HOMA-IR and negative correlation with HOMA-β. The m6A methylated RNA and METTL3 levels were also up-regulated in mouse with 16 weeks high-fat diet (HFD), compared with mice fed a standard chow diet (CD). Hepatocyte-specific knockout of METTL3 in mice fed a HFD improved insulin sensitivity and decreased fatty acid synthesis. Furthermore, mechanism analysis demonstrates that METTL3 silence decreased the m6A methylated and total mRNA level of Fatty acid synthase (Fasn), subsequently inhibited fatty acid metabolism. Adeno-associated virus mediated Fasn overexpression in METTL3 knockout mice abrogates the improved insulin sensitivity and decreased fatty acid synthesis. Collectively, these results reveal that RNA N6-methyladenosine methylase METTL3 inhibits hepatic insulin sensitivity via N6-methylation of Fasn mRNA and promoting fatty acid metabolism. 10.1016/j.bbrc.2019.08.018
    FTO modifies the m6A level of MALAT and promotes bladder cancer progression. Tao Le,Mu Xingyu,Chen Haige,Jin Di,Zhang Ruiyun,Zhao Yuyang,Fan Jie,Cao Ming,Zhou Zhihua Clinical and translational medicine BACKGROUND:Nearly a half million people around the world are diagnosed with bladder cancer each year, and an incomplete understanding of its pathogenicity and lack of efficient biomarkers having been discovered lead to poor clinical management of bladder cancer. Fat mass and obesity-associated protein (FTO) is a critical player in carcinogenesis. We, here, explored the role of FTO and unraveled the mechanism of its function in bladder cancer. METHODS:Identification of the correlation of FTO with bladder cancer was based on both bioinformatics and clinical analysis of tissue samples collected from a cohort of patients at a hospital and microarray data. Gain-of-function and loss-of-function assays were conducted in vivo and in vitro to assess the effect of FTO on bladder carcinoma tumor growth and its impact on the bladder carcinoma cell viability. Moreover, the interactions of intermediate products were also investigated to elucidate the mechanisms of FTO function. RESULTS:Bladder tumor tissues had increased FTO expression which correlated with clinical bladder cancer prognosis and outcomes. Both in vivo and in vitro, it played the function of an oncogene in stimulating the cell viability and tumorigenicity of bladder cancer. Furthermore, FTO catalyzed metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) demethylation, regulated microRNA miR-384 and mal T cell differentiation protein 2 (MAL2) expression, and modulated the interactions among these processes. CONCLUSIONS:The interplay of these four clinically relevant factors contributes to the oncogenesis of bladder cancer. FTO facilitates the tumorigenesis of bladder cancer through regulating the MALAT/miR-384/MAL2 axis in m6A RNA modification manner, which ensures the potential of FTO for serving as a diagnostic or prognostic biomarker in bladder cancer. 10.1002/ctm2.310
    The Complex Roles and Therapeutic Implications of mA Modifications in Breast Cancer. Wei Min,Bai Jing-Wen,Niu Lei,Zhang Yong-Qu,Chen Hong-Yu,Zhang Guo-Jun Frontiers in cell and developmental biology Accumulating evidence indicates that N-methyladenosine (mA), which directly regulates mRNA, is closely related to multiple biological processes and the progression of different malignancies, including breast cancer (BC). Studies of the aberrant expression of mA mediators in BC revealed that they were associated with different BC subtypes and functions, such as proliferation, apoptosis, stemness, the cell cycle, migration, and metastasis, through several factors and signaling pathways, such as Bcl-2 and the PI3K/Akt pathway, among others. Several regulators that target mA have been shown to have anticancer effects. Fat mass and obesity-associated protein (FTO) was identified as the first mA demethylase, and a series of inhibitors that target FTO were reported to have potential for the treatment of BC by inhibiting cell proliferation and promoting apoptosis. However, the exact mechanism by which mA modifications are regulated by FTO inhibitors remains unknown. mA modifications in BC have only been preliminarily studied, and their mechanisms require further investigation. 10.3389/fcell.2020.615071
    mA demethylase FTO facilitates tumor progression in lung squamous cell carcinoma by regulating MZF1 expression. Liu Jiqin,Ren Dangli,Du Zhenhua,Wang Hekong,Zhang Hua,Jin Ying Biochemical and biophysical research communications N-Methyladenosine (mA) represents the most prevalent internal modification in mammalian mRNAs. Emerging evidences suggest that mA modification is profoundly implicated in many biological processes, including cancer development. However, limited knowledge is available about the functional importance of mA in lung cancer. In this study, by data mining The Cancer Genome Atlas (TCGA) database, we first identified fat mass- and obesity-associated protein (FTO) as a prognostic factor for lung squamous cell carcinoma (LUSC). Then we showed that FTO, but not other mA modification genes including METTL3, METTL14 and ALKBH5, was the major dysregulated factor responsible for aberrant mA modification in LUSC. Loss-of-function studies suggested that FTO knockdown effectively inhibited cell proliferation and invasion, while promoted cell apoptosis of L78 and NCI-H520 cells. Furthermore, overexpression of FTO, but not its mutant form, facilitated the malignant phenotypes of CHLH-1 cells. Mechanistically, FTO enhanced MZF1 expression by reducing mA levels and mRNA stability in MZF1 mRNA transcript, leading to oncogenic functions. Taken together, our study demonstrates the functional importance of FTO in the tumor progression of LUSC and provides a potential therapeutic target for LUSC treatment. 10.1016/j.bbrc.2018.05.175
    FTO regulates myoblast proliferation by controlling CCND1 expression in an mA-YTHDF2-dependent manner. Deng Kaiping,Zhang Zhen,Ren Caifang,Liang Yaxu,Gao Xiaoxiao,Fan Yixuan,Wang Feng Experimental cell research N6-Methyladenosine (mA) modification is the most abundant chemical modification in mRNA, and it participates in various biological processes, such as cell differentiation and proliferation. However, little is known about the function of mA demethylase fat mass and obesity-associated (FTO) in myoblast proliferation. Here, we demonstrated that knockdown of FTO can significantly inhibit myoblast proliferation and promote apoptosis. RNA sequencing analysis revealed that a lot of downregulated genes in FTO knockdown cells are associated with cell cycle and apoptosis. Furthermore, silencing FTO drastically decreased cyclin D1 (CCND1) expression through YTHDF2-mediated mRNA degradation, thereby delaying the progression of G1 phase, and leading to impaired myoblast proliferation. These findings unraveled that FTO regulates myoblast proliferation by controlling CCND1 expression in an mA-YTHDF2-dependent manner, which highlights the critical roles of mA modification in myoblast proliferation. 10.1016/j.yexcr.2021.112524
    MTCH2 promotes adipogenesis in intramuscular preadipocytes via an mA-YTHDF1-dependent mechanism. Jiang Qin,Sun Baofa,Liu Qing,Cai Min,Wu Ruifan,Wang Fengqin,Yao Yongxi,Wang Yizhen,Wang Xinxia FASEB journal : official publication of the Federation of American Societies for Experimental Biology Intramuscular fat is considered a potential factor that is associated with meat quality in animal production and insulin resistance in humans. N-methyladenosine (mA) modification of mRNA plays an important role in regulating adipogenesis. However, the effects of mA on the adipogenesis of intramuscular preadipocytes and associated mechanisms remain unknown. Here, we performed mA sequencing to compare mA methylome of the longissimus dorsi muscles (LDMs) between Landrace pigs (lean-type breed) and Jinhua pigs (obese-type breed with higher levels of intramuscular fat). Transcriptome-wide mA profiling of porcine LDMs was highly conserved with humans and mice. Furthermore, we identified a unique methylated gene in Jinhua pigs named mitochondrial carrier homology 2 ( MTCH2). The mA levels of MTCH2 mRNA were reduced by introducing a synonymous mutation, and adipogenesis test results showed that the MTCH2 mutant was inferior with regard to adipogenesis compared with the MTCH2 wild-type. We then found that MTCH2 protein expression was positively associated with mA levels, and an YTH domain family protein 1-RNA immunoprecipitation-quantitative PCR assay indicated that MTCH2 mRNA was a target of the YTH domain family protein 1. This study provides comprehensive mA profiles of LDM transcriptomes in pigs and suggests an essential role for mA modification of MTCH2 in intramuscular fat regulation.-Jiang, Q., Sun, B., Liu, Q., Cai, M., Wu, R., Wang, F., Yao, Y., Wang, Y., Wang, X. MTCH2 promotes adipogenesis in intramuscular preadipocytes via an mA-YTHDF1-dependent mechanism. 10.1096/fj.201801393RRR
    mA mRNA methylation regulates testosterone synthesis through modulating autophagy in Leydig cells. Chen Yabing,Wang Jing,Xu Dihui,Xiang Zou,Ding Jie,Yang Xiaoyu,Li Dongmei,Han Xiaodong Autophagy Macroautophagy/autophagy is indispensable for testosterone synthesis in Leydig cells (LCs), and here we report a negative association between mA modification and autophagy in LCs during testosterone synthesis. A gradual decrease of METTL14 (methyltransferase like 14) and an increase of ALKBH5 (alkB homolog 5, RNA demethylase) were observed in LCs during their differentiation from stem LCs to adult LCs. These events led to reduced mRNA methylation levels of N-methyladenosine (mA) and enhanced autophagy in LCs. Similar regulation of METTL14, ALKBH5, and mA was also observed in LCs upon treatment with human chorionic gonadotropin (HsCG). Mechanistically, mA modification promoted translation of PPM1A (protein phosphatase 1A, magnesium dependent, alpha isoform), a negative AMP-activated protein kinase (AMPK) regulator, but decreased expression of CAMKK2 (calcium/calmodulin-dependent protein kinase kinase 2, beta), a positive AMPK regulator, by reducing its RNA stability. Thus, mA modification resulted in reduced AMPK activity and subsequent autophagy inhibition. We further demonstrated that ALKBH5 upregulation by HsCG was dependent on enhanced binding of the transcriptional factor CEBPB (CCAAT/enhancer binding protein [C/EBP], beta) and the TFEB (transcription factor EB) to its gene promoter. Moreover, HsCG treatment decreased METTL14 by reducing its stability. Collectively, this study highlights a vital role of mA RNA methylation in the modulation of testosterone synthesis in LCs, providing insight into novel therapeutic strategies by exploiting mA RNA methylation as targets for treating azoospermatism and oligospermatism patients with reduction in serum testosterone. 3-MA: 3-methyladenine; ACTB: Actin, beta; ALKBH5: alkB homolog 5, RNA demethylase; AMPK: AMP-activated protein kinase; BafA1: bafilomycin A CAMKK2: calcium/calmodulin-dependent protein kinase kinase 2, beta; CEBPB: CCAAT/enhancer-binding protein (C/EBP), beta; ChIP: chromatin immunoprecipitation; FTO: fat mass and obesity associated; HsCG: human chorionic gonadotropin; HSD3B: 3β-hydroxysteroid dehydrogenase; LCs: Leydig cells; mA: N-methyladenosine; METTL14: methyltransferase like 14; METTL3: methyltransferase like 3; MTOR: mechanistic target of rapamycin kinase; PPM1A: protein phosphatase 1A, magnesium dependent, alpha isoform; PRKAA: 5'-AMP-activated protein kinase catalytic subunit alpha; SQSTM1: sequestosome 1; STK11/LKB1: serine/threonine kinase 11; TFEB: transcription factor EB; ULK1: unc-51-like kinase 1; WTAP: Wilms tumor 1-associating protein; YTHDF: YTH N6-methyladenosine RNA binding protein. 10.1080/15548627.2020.1720431
    Hepatic expression of FTO and fatty acid metabolic genes changes in response to lipopolysaccharide with alterations in mA modification of relevant mRNAs in the chicken. Zhang Y,Guo F,Zhao R British poultry science The fat mass and obesity associated (FTO) gene, which encodes a demethylase of mA, has been reported to respond to lipopolysaccharide (LPS) and to serve as a link between inflammation and metabolic responses. The objective of this study was to determine whether LPS-induced changes in the expression of FTO and metabolic genes are associated with alterations of mA in relevant mRNAs. LPS challenge significantly decreased hepatic mRNA expression of carnitine palmitoyl transferase 1 (CPT1) and CPT2, which coincided with a tendency of higher triglyceride accumulation in the liver. LPS significantly down-regulated the full length cFTO1, yet up-regulated the truncated cFTO4 protein in the liver nuclear extracts. Nuclear protein content of cFTO4 in the liver was negatively correlated with the mRNA abundances of CPT1 (r = 0.629) and CPT2 (r = 0.622). Methylated RNA immunoprecipitation analysis revealed that the mA level around the translation start site of CPT1 was markably decreased in the liver of LPS-treated chickens. These results indicate that LPS-induced changes in FTO protein expression are associated with alteration of mRNA mA modification in chicken liver. 10.1080/00071668.2016.1201199
    mA RNA Methylation Controls Neural Development and Is Involved in Human Diseases. Du Kunzhao,Zhang Longbin,Lee Trevor,Sun Tao Molecular neurobiology RNA modifications are involved in many aspects of biological functions. N6-methyladenosine (mA) is one of the most important forms of RNA methylation and plays a vital role in regulating gene expression, protein translation, cell behaviors, and physiological conditions in many species, including humans. The dynamic and reversible modification of mA is conducted by three elements: methyltransferases ("writers"), such as methyltransferase-like protein 3 (METTL3) and METTL14; mA-binding proteins ("readers"), such as the YTH domain family proteins (YTHDFs) and YTH domain-containing protein 1 (YTHDC1); and demethylases ("erasers"), such as fat mass and obesity-associated protein (FTO) and AlkB homolog 5 (ALKBH5). In this review, we summarize the current knowledge on mapping mRNA positions of mA modification and revealing molecular processes of mA. We further highlight the biological significance of mA modification in neural cells during development of the nervous system and its association with human diseases. mA RNA methylation is becoming a new frontier in neuroscience and should help us better understand neural development and neurological diseases from a novel point of view. 10.1007/s12035-018-1138-1
    Ling-gui-zhu-gan decoction alleviates hepatic steatosis through SOCS2 modification by N6-methyladenosine. Dang Yanqi,Xu Jingjuan,Yang Yang,Li Chunlin,Zhang Qiang,Zhou Wenjun,Zhang Li,Ji Guang Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie BACKGROUND:The ling-gui-zhu-gan (LGZG) decoction is a classic formula in traditional chinese medicine (TCM) and is widely used in clinical settings. Recently, the LGZG decoction was demonstrated to have an effect in alleviating hepatic steatosis induced by a high-fat diet (HFD). However, the mechanisms underlying this therapeutic effect remain unclear. The present study was designed to evaluate the effect and explore possible mechanisms of action of the LGZG decoction in nonalcoholic fatty liver disease (NAFLD). METHODS:Liver tissue and blood samples were harvested. Liver steatosis, triglyceride (TG), liver total cholesterol (TC), liver low-density lipoprotein (LDL), serum almandine aminotransferase (ALT), aspartate aminotransferase (AST), and free fatty acid (FFA) were assayed. N6-methyladenosine (m6A) levels were estimated using an m6A RNA methylation quantification kit and immunohistochemistry. The m6A methylome was detected through methylated RNA immunoprecipitation sequencing (MeRIP-seq), followed by data analysis. The expression levels of differentially methylated genes (DMGs) were determined using real-time polymerase chain reaction and western blotting. RESULTS:The LGZG decoction significantly alleviated hepatic steatosis and reduced m6A levels. MeRIP-seq revealed the coding sequence (CDS) domain to be the most critical modification site for m6A methylation, and the molecular functions of DMGs predominantly included insulin-like growth factor receptor binding and fatty acid metabolism and degradation. Further, LGZG treatment could reduce the m6A methylation levels of suppressor of cytokine signaling 2 (SOCS2), along with the expression of SOCS2 at mRNA and protein levels. CONCLUSIONS:The LGZG decoction is an effective formula for treating NAFLD, and the possible mechanisms underlying its action could be related to N6-methyladenosine modification-medicated SOCS2. 10.1016/j.biopha.2020.109976
    Profiling of RNA -Methyladenosine Methylation Reveals the Critical Role of mA in Chicken Adipose Deposition. Cheng Bohan,Leng Li,Li Ziwei,Wang Weijia,Jing Yang,Li Yudong,Wang Ning,Li Hui,Wang Shouzhi Frontiers in cell and developmental biology One of the main objectives of broiler breeding is to prevent excessive abdominal adipose deposition. The role of RNA modification in adipose deposition is not clear. This study was aimed to map mA modification landscape in chicken adipose tissue. MeRIP-seq was performed to compare the differences in mA methylation pattern between fat and lean broilers. We found that start codons, stop codons, coding regions, and 3'-untranslated regions were generally enriched for mA peaks. The high mA methylated genes (fat birds vs. lean birds) were primarily associated with fatty acid biosynthesis and fatty acid metabolism, while the low mA methylated genes were mainly involved in processes associated with development. Furthermore, we found that the mRNA levels of many genes may be regulated by mA modification. This is the first comprehensive characterization of mA patterns in the chicken adipose transcriptome, and provides a basis for studying the role of mA modification in fat deposition. 10.3389/fcell.2021.590468
    FTO demethylates m6A modifications in HOXB13 mRNA and promotes endometrial cancer metastasis by activating the WNT signalling pathway. Zhang Lin,Wan Yicong,Zhang Zihan,Jiang Yi,Lang Jinghe,Cheng Wenjun,Zhu Lan RNA biology Although many studies have confirmed the relationship between obesity and endometrial cancer (EC), the molecular mechanism between obesity and EC progression has not been elucidated. Overexpression of fat mass and the obesity associated protein FTO leads to weight gain, although recently it has been discovered that FTO can serve as a demethylase which erases N6-methyladenosine (m6A) modification and regulates the metabolization of mRNAs. In this study, we found high expression of FTO in metastatic EC and that this action promote both metastasis and invasion in vivo and in vitro. Mechanistically, FTO can catalyse demethylation modification in 3'UTR region of HOXB13 mRNA, thereby abolishing m6A modification recognition with the YTHDF2 protein. Decreasing HOXB13 mRNA decay and increasing HOXB13 protein expression was accompanied by WNT signalling pathway activation and the expression of downstream proteins, leading to tumour metastasis and invasion. We also found the WNT signalling pathway inhibitor ICG-001 can block HOXB13 gene-induced tumour metastasis, therefore ICG-001 may be a promising molecular intervention. This study provides insight into the relationship between obesity and the pathogenesis of endometrial cancer while highlighting future areas of research. 10.1080/15476286.2020.1841458
    Novel positioning from obesity to cancer: FTO, an mA RNA demethylase, regulates tumour progression. Chen JiaLing,Du Bin Journal of cancer research and clinical oncology PURPOSE:The fat mass- and obesity-associated (FTO) gene on chromosome 16q12.2 shows an intimate association with obesity and body mass index. Recently, research into the FTO gene and its expression product has attracted widespread interest due to the identification of FTO as an N6-methyladenosine (m6A) demethylase. FTO primarily regulates the m6A levels of downstream targets via their 3' untranslated regions. FTO not only plays a critical role in obesity-related diseases but also is involved in the occurrence, development and prognosis of many types of cancer, such as acute myeloid leukaemia, glioblastoma and breast cancer. Currently, studies indicate that FTO is a crucial component of m6A modification, it regulates cancer stem cell function, and promotes the growth, self-renewal and metastasis of cancer cells. In this review, we summarized and analysed the data regarding the structural features and biological functions of FTO as well as its association with different cancers and possible molecular mechanisms. METHODS:We systematically reviewed the related literatures regarding FTO and its demethylation activity in many pathologic and physiological processes, especially in cancer-related diseases based on PubMed databases in this article. RESULTS:Mounting evidence indicated that FTO plays a critical role in occurrence, progression and treatment of various cancers, even acting as a cancer oncogene in acute myeloid leukaemia, research on which is no longer restricted to metabolic diseases such as obesity and diabetes. CONCLUSION:Considering FTO's critical role in many diseases, FTO may become a new promising target for the diagnosis and treatment of various diseases in the near future, especially for specific types of cancers, such as acute myeloid leukaemia, glioblastoma and breast cancer. 10.1007/s00432-018-2796-0
    m6A demethylase FTO promotes hepatocellular carcinoma tumorigenesis via mediating PKM2 demethylation. Li Jie,Zhu Lijun,Shi Yanhong,Liu Jingnan,Lin Lin,Chen Xi American journal of translational research N6-methyladenosine (m6A) acts as the most common mRNA modification in mammal cells. Fat Mass and Obesity-associated protein (FTO) is the firstly identified demethylase in the m6A modification. This research tries to discover the potential roles of FTO in the m6A modification in the hepatocellular carcinoma (HCC). FTO level was found to be up-regulated in the HCC tissue and cells. The over-expression of FTO was correlated to the poor prognosis of HCC individuals. Knockdown of FTO suppressed the proliferation and in vivo tumor growth, and induced the G0/G1 phase arrest. Mechanically, FTO triggered the demethylation of PKM2 mRNA and accelerated the translated production. Overall, this finding suggests the critical function of FTO in the HCC oncogenesis and its m6A modification.
    Early-Life mA RNA Demethylation by Fat Mass and Obesity-Associated Protein (FTO) Influences Resilience or Vulnerability to Heat Stress Later in Life. Kisliouk Tatiana,Rosenberg Tali,Ben-Nun Osher,Ruzal Mark,Meiri Noam eNeuro Early life heat stress leads to either resilience or vulnerability to a similar stress later in life. We have previously shown that this tuning of the stress response depends on neural network organization in the preoptic anterior hypothalamus (PO/AH) thermal response center and is regulated by epigenetic mechanisms. Here, we expand our understanding of stress response establishment describing a role for epitranscriptomic regulation of the epigenetic machinery. Specifically, we explore the role of N-methyladenosine (mA) RNA methylation in long-term response to heat stress. Heat conditioning of 3-d-old chicks diminished mA RNA methylation in the hypothalamus, simultaneously with an increase in the mRNA levels of the mA demethylase, fat mass and obesity-associated protein (). Moreover, a week later, methylation of two heat stress-related transcripts, histone 3 lysine 27 (H3K27) methyltransferase, enhancer of zeste homolog 2 () and brain-derived neurotrophic factor (), were downregulated in harsh-heat-conditioned chicks. During heat challenge a week after conditioning, there was a reduction of mA levels in mild-heat-conditioned chicks and an elevation in harsh-heat-conditioned ones. This increase in mA modification was negatively correlated with the expression levels of both and Antisense "knock-down" of FTO caused an elevation of global mA RNA methylation, reduction of and mRNA levels, and decrease in global H3K27 dimethylation as well as dimethyl H3K27 level along coding region, and, finally, led to heat vulnerability. These findings emphasize the multilevel regulation of gene expression, including both epigenetic and epitranscriptomic regulatory mechanisms, fine-tuning the neural network organization in a response to stress. 10.1523/ENEURO.0549-19.2020
    m6A Demethylase FTO Regulates Dopaminergic Neurotransmission Deficits Caused by Arsenite. Bai LuLu,Tang Qianghu,Zou Zhen,Meng Pan,Tu Baijie,Xia Yinyin,Cheng Shuqun,Zhang Lina,Yang Kai,Mu Shaoyu,Wang Xuefeng,Qin Xia,Lv Bo,Cao Xianqing,Qin Qizhong,Jiang Xuejun,Chen Chengzhi Toxicological sciences : an official journal of the Society of Toxicology Arsenite exposure is known to increase the risk of neurological disorders via alteration of dopamine content, but the detailed molecular mechanisms remain largely unknown. In this study, using both dopaminergic neurons of the PC-12 cell line and C57BL/6J mice as in vitro and in vivo models, our results demonstrated that 6 months of arsenite exposure via drinking water caused significant learning and memory impairment, anxiety-like behavior and alterations in conditioned avoidance and escape responses in male adult mice. We also were the first to reveal that the reduction in dopamine content induced by arsenite mainly resulted from deficits in dopaminergic neurotransmission in the synaptic cleft. The reversible N6- methyladenosine (m6A) modification is a novel epigenetic marker with broad roles in fundamental biological processes. We further evaluated the effect of arsenite on the m6A modification and tested if regulation of the m6A modification by demethylase fat mass and obesity-associated (FTO) could affect dopaminergic neurotransmission. Our data demonstrated for the first time that arsenite remarkably increased m6A modification, and FTO possessed the ability to alleviate the deficits in dopaminergic neurotransmission in response to arsenite exposure. Our findings not only provide valuable insight into the molecular neurotoxic pathogenesis of arsenite exposure, but are also the first evidence that regulation of FTO may be considered as a novel strategy for the prevention of arsenite-associated neurological disorders. 10.1093/toxsci/kfy172
    Meclofenamic acid represses spermatogonial proliferation through modulating mA RNA modification. Huang Tao,Guo Jiayin,Lv Yinghua,Zheng Yi,Feng Tongying,Gao Qiang,Zeng Wenxian Journal of animal science and biotechnology Background:N6-Methyladenosine (mA), the most prevalent modification in mammalian mRNA, plays important roles in numerous biological processes. Several mA associated proteins such as methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) and YTH domain containing 2 (YTHDC2) are involved in the regulation of spermatogenesis and oogenesis. However, the role of the first detected m6A demethylase, fat mass and obesity associate protein (FTO), in germ cells remains elusive. Elucidation of FTO roles in the regulation of germ cell fate will provide novel insights into the mammalian reproduction. Methods:Mouse GC-1 spg cells were treated with the ester form of meclofenamic acid (MA2) to inhibit the demethylase activity of FTO. The cellular mA and mA level were analyzed through high performance liquid chromatography combined with tandem mass spectrometry (HPLC/MS-MS). The cell apoptosis was detected via TUNEL and flow cytometry. The cell proliferation was detected through EdU and western blot. The mRNA level of core cyclin dependent kinases (CDKs) was quantified via q-PCR. RNA decay assay were performed to detect RNA stability. Dual fluorescence assay was conducted to study whether MA2 affects the expression of CDK2 dependent on the mA modification at 3'UTR. Results:MA2 significantly increased the cellular mA level and down-regulated the expression of CDK1, CDK2, CDK6 and CdC25a, resulting in arrest of G1/S transition and decrease of cell proliferation. MA2 downregulated CDK2 mRNA stability. Additionally, mutation of the predicted mA sites in the Cdk2-3'UTR could mitigated the degradation of CDK2 mRNA after MA2 treatment. Conclusion:MA2 affected CDKs expression through the mA-dependent mRNA degradation pathway, and thus repressed spermatogonial proliferation. 10.1186/s40104-019-0361-6
    Structural and Virus Regulatory Insights Into Avian N6-Methyladenosine (m6A) Machinery. Bayoumi Mahmoud,Rohaim Mohammed A,Munir Muhammad Frontiers in cell and developmental biology The addition of a methyl group to the N6 position of adenosine (m6A) is the most common posttranscriptional RNA modification, and it regulates most steps of RNA metabolism including splicing, stability, translation, nuclear-export, and RNA structures. Besides cellular RNA, m6A modifications have also been detected on viral RNA. A range of recent studies have demonstrated the crucial roles of m6A in the virus-host interactions; however, m6A cellular machineries are only characterized in limited mammalian species. Herein, we aim to present comprehensive evolutionary insights into major m6A writers, erasers, and readers and draw a comparative structural analysis between avian and mammalian m6A-associated machineries. The comparative collinearity on the chromosomal scale revealed that the majority of m6A-related genes were found less syntenic even among avian species. Genetic analysis of avian m6A erasers revealed a distinct phylogenetic clustering compared to mammalian orthologs and shared a weak percent (55%) identity with mammalian species with low identity percentage (55%). The overall comparative three-dimensional (3D) structure analyses among different mammalian species were maintained through synonymous structural mutations. Unlike erasers, the putative 3D structures in the active sites as for the aromatic cage in YTH-domain of YTHDC1 and two pivotal loops in MTD-domains in METTL3 exhibited structural alterations in chicken. In conjunction with investigations, influenza viruses significantly downregulated gene the transcription of m6A writers and erasers, whereas m6A readers were moderately regulated in chicken fibroblasts. In light of these findings, future detailed biochemical and crystallographic studies are warranted to define the roles of m6A machinery in regulating both viral and cellular RNA metabolism in avian species. 10.3389/fcell.2020.00543
    Resveratrol Attenuates High-Fat Diet Induced Hepatic Lipid Homeostasis Disorder and Decreases mA RNA Methylation. Wu Jiamin,Li Yi,Yu Jiayao,Gan Zhending,Wei Wenyao,Wang Chao,Zhang Lili,Wang Tian,Zhong Xiang Frontiers in pharmacology -methyladenosine (mA) mRNA methylation is affected by dietary factors and associated with lipid metabolism; however, whether the regulatory role of resveratrol in lipid metabolism is involved in mA mRNA methylation remains unknown. Here, the objective of this study was to investigate the effect of resveratrol on hepatic lipid metabolism and mA RNA methylation in the liver of mice. A total of 24 male mice were randomly allocated to LFD (low-fat diet), LFDR (low-fat diet + resveratrol), HFD (high-fat diet), and HFDR (high-fat diet + resveratrol) groups for 12 weeks ( = 6/group). Final body weight of mice was measured before sacrificing. Perirhemtric fat, abdominal and epididymal fat, liver tissues, and serum were collected at sacrifice and analyzed. Briefly, mice phenotype, lipid metabolic index, and mA modification in the liver were assessed. Compared to the HFD group, dietary resveratrol supplementation reduced the body weight and relative abdominal, epididymal, and perirhemtric fat weight in high-fat-exposed mice; however, resveratrol significantly increased average daily feed intake in mice given HFD. The amounts of serum low-density lipoprotein cholesterol (LDL), liver total cholesterol (TC), and triacylglycerol (TAG) were significantly decreased by resveratrol supplementation. In addition, resveratrol significantly enhanced the levels of peroxisome proliferator-activated receptor alpha (), peroxisome proliferator-activated receptor beta/delta (), cytochrome P450, family 4, subfamily a, polypeptide 10/14 (/), acyl-CoA oxidase 1 (), and fatty acid-binding protein 4 () mRNA and inhibited acyl-CoA carboxylase () mRNA levels in the liver. Furthermore, the resveratrol in HFD increased the transcript levels of methyltransferase like 3 (), alkB homolog 5 (), fat mass and obesity associated protein (), and YTH domain family 2 (), whereas it decreased the level of YTH domain family 3 () and mA abundance in mice liver. The beneficial effect of resveratrol on lipid metabolism disorder under HFD may be due to decrease of mA RNA methylation and increase of mRNA, providing mechanistic insights into the function of resveratrol in alleviating the disturbance of lipid metabolism in mice. 10.3389/fphar.2020.568006
    Role of m6A RNA methylation in cardiovascular disease (Review). Qin Yuhan,Li Linqing,Luo Erfei,Hou Jiantong,Yan Gaoliang,Wang Dong,Qiao Yong,Tang Chengchun International journal of molecular medicine N6‑methyladenosine (m6A) is the most prevalent and abundant type of internal post‑transcriptional RNA modification in eukaryotic cells. Multiple types of RNA, including mRNAs, rRNAs, tRNAs, long non‑coding RNAs and microRNAs, are involved in m6A methylation. The biological function of m6A modification is dynamically and reversibly mediated by methyltransferases (writers), demethylases (erasers) and m6A binding proteins (readers). The methyltransferase complex is responsible for the catalyzation of m6A modification and is typically made up of methyltransferase‑like (METTL)3, METTL14 and Wilms tumor 1‑associated protein. Erasers remove methylation by fat mass and obesity‑associated protein and ALKB homolog 5. Readers play a role through the recognition of m6A‑modified targeted RNA. The YT521‑B homology domain family, heterogeneous nuclear ribonucleoprotein and insulin‑like growth factor 2 mRNA‑binding protein serve as m6A readers. The m6A methylation on transcripts plays a pivotal role in the regulation of downstream molecular events and biological functions, such as RNA splicing, transport, stability and translatability at the post‑transcriptional level. The dysregulation of m6A modification is associated with cancer, drug resistance, virus replication and the pluripotency of embryonic stem cells. Recently, a number of studies have identified aberrant m6A methylation in cardiovascular diseases (CVDs), including cardiac hypertrophy, heart failure, arterial aneurysm, vascular calcification and pulmonary hypertension. The aim of the present review article was to summarize the recent research progress on the role of m6A modification in CVD and give a brief perspective on its prospective applications in CVD. 10.3892/ijmm.2020.4746
    Increased m6A modification of RNA methylation related to the inhibition of demethylase FTO contributes to MEHP-induced Leydig cell injury. Zhao Tianxin,Wang Junke,Wu Yuhao,Han Lindong,Chen Jiadong,Wei Yuexin,Shen Lianju,Long Chunlan,Wu Shengde,Wei Guanghui Environmental pollution (Barking, Essex : 1987) N-methyladenosine (m6A) modification, the most prevalent form of RNA methylation, modulates gene expression post-transcriptionally. Di-(2-ethylhexyl) phthalate (DEHP) is a common environmental endocrine disrupting chemical that induces testicular injury due to the inhibition of the demethylase fat mass and obesity-associated protein (FTO) and increases the m6A modification. How FTO-mediated m6A modification in testicular Leydig cell injury induced by DEHP remains unclear. Here, the TM3 Leydig cell line was treated with mono-(2-ethylhexyl) phthalate (MEHP), the main metabolite of DEHP in the body, as well as FB23-2, an inhibitor of FTO. Decreased levels of testosterone in the culture supernatant, significantly increased apoptosis, and a remarkable upregulation of global m6A modification were found in both TM3 cells treated with MEHP and FB23-2. Transcriptome sequencing showed that both treatments significantly induced apoptosis-associated gene expression. Methylated RNA immunoprecipitation sequencing showed that the Leydig cell injury induced by upregulated m6A modification could be associated with multiple physiological disorders, including histone acetylation, reactive oxygen species biosynthesis, MAPK signaling pathway, hormone secretion regulation, autophagy regulation, and male gonadal development. Overall, the inhibition of FTO-mediated up-regulation of m6A could be involved in MEHP-induced Leydig cell apoptosis. 10.1016/j.envpol.2020.115627
    Excessive BCAA regulates fat metabolism partially through the modification of mA RNA methylation in weanling piglets. Heng Jinghui,Wu Zhihui,Tian Min,Chen Jiaming,Song Hanqing,Chen Fang,Guan Wutai,Zhang Shihai Nutrition & metabolism Background:Fat percentage and distribution in pigs are associated with their productive efficiency and meat quality. Dietary branched-chain amino acids (BCAA) regulate fat metabolism in weanling piglets with unknown mechanism. It is reported that N6-methyl-adenosine (mA) is involved in fat metabolism in mice. The current study was designed to investigate the relationship between dietary branched-chain amino acids and fat metabolism through N6-methyl-adenosine (mA) in weanling piglets. Methods:A total of 18 healthy crossbred weaned piglets (Duroc × Landrace × Large White, 10.45 ± 0.41 kg) were divided into 3 treatments and were fed the low BCAA dose diet (L-BCAA), the normal dose BCAA diet (N-BCAA), or the high dose BCAA (H-BCAA) diet for 3 weeks. Results:Our results show that compared with the N-BCAA group, the L-BCAA group had higher concentration of serum leptin ( < 0.05), while the H-BCAA group had lower concentration of serum adiponectin ( < 0.05). Fatty acid synthesis in pigs from the H-BCAA group was lower than those from the N-BCAA group with the down-regulation of lipogenic genes (ACACA, FASN, PPAR-r, SREBP-1c in ventral and dorsal fat, SREBP-1c in liver) and up-regulation of lipolysis genes (HSL, ATGL, CPT-1A, FABP4 in ventral fat, HSL in liver) ( < 0.05). Similarly, fatty acid synthesis in pigs from the L-BCAA group was also lower than those from the N-BCAA group with the decrease of lipogenic genes (ACACA in ventral, ACACA and FASN in dorsal fat, ACACA, FASN, SREBP-1c in liver) and the increase of lipolysis genes (ATGL, CPT-1A CD36, FABP4 in ventral fat and HSL, ATGL, CPT-1A in dorsal fat, CPT-1A) ( < 0.05). Feeding H-BCAA diet significantly reduced total mA levels in ventral and dorsal fat and liver tissues ( < 0.05). The decrease of total mA is associated with down-regulation of METTL3, METTL14 and FTO in dorsal fat and METTL3 and FTO in liver ( < 0.05). Decreased mA modification of ACACA and FASN in ventral and dorsal adipose tissues was observed in pig fed with excessive BCAA. Conclusion:These results suggest that insufficient or excessive BCAA decreased the fat deposition by increasing lipolysis and deceasing lipogenesis in adipose and liver tissues. Dietary excessive BCAA might regulate the process of lipid metabolism partly through the mA RNA methylation. 10.1186/s12986-019-0424-x