Novel insights into the roles of RNA N-methyladenosine modification in regulating gene expression during environmental exposures. Li Dong,Zhu Xiaohua,Li Yunxiang,Zeng Xianyin Chemosphere N-methyladenosine (mA) is one of the most common RNA modifications in eukaryotes involved in the regulation of post-transcriptional gene expression, as well as the occurrence and development of diseases related to environmental exposures. Adverse factors produced by environmental exposures, such as reactive oxygen species, inflammation, and cyclobutane pyrimidine dimers, mediate mA modification, thereby regulating downstream gene and protein expression, and signaling pathways, such as FTO/mA RNA/p53 axis, PI3K/AKT/mTOR pathway, and PARP/METTL3/mA RNA/Pol κ pathway. Moreover, an imbalance in mA methylation levels directly mediates disease pathogenesis. To date, some studies have detailed the mechanisms underlying environmental exposure-mediated global changes in RNA mA methylation. Based on our current understanding, we aimed to elaborate on the molecular mechanisms through which RNA mA methylation regulates gene expression under environmental exposures. In this review, we outline the biogenesis and functions of RNA mA modification. Furthermore, we focus on the effects of environmental exposures on mA levels and highlight the relationships between environmental exposures (doses and time) and mA levels. Although the molecular mechanisms regulating gene expression remains to be elucidated, mA has potential applications as a disease biomarker. 10.1016/j.chemosphere.2020.127757
    Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Dominissini Dan,Moshitch-Moshkovitz Sharon,Schwartz Schraga,Salmon-Divon Mali,Ungar Lior,Osenberg Sivan,Cesarkas Karen,Jacob-Hirsch Jasmine,Amariglio Ninette,Kupiec Martin,Sorek Rotem,Rechavi Gideon Nature An extensive repertoire of modifications is known to underlie the versatile coding, structural and catalytic functions of RNA, but it remains largely uncharted territory. Although biochemical studies indicate that N(6)-methyladenosine (m(6)A) is the most prevalent internal modification in messenger RNA, an in-depth study of its distribution and functions has been impeded by a lack of robust analytical methods. Here we present the human and mouse m(6)A modification landscape in a transcriptome-wide manner, using a novel approach, m(6)A-seq, based on antibody-mediated capture and massively parallel sequencing. We identify over 12,000 m(6)A sites characterized by a typical consensus in the transcripts of more than 7,000 human genes. Sites preferentially appear in two distinct landmarks--around stop codons and within long internal exons--and are highly conserved between human and mouse. Although most sites are well preserved across normal and cancerous tissues and in response to various stimuli, a subset of stimulus-dependent, dynamically modulated sites is identified. Silencing the m(6)A methyltransferase significantly affects gene expression and alternative splicing patterns, resulting in modulation of the p53 (also known as TP53) signalling pathway and apoptosis. Our findings therefore suggest that RNA decoration by m(6)A has a fundamental role in regulation of gene expression. 10.1038/nature11112
    Post-transcriptional regulation by the exosome complex is required for cell survival and forebrain development via repression of P53 signaling. Ulmke Pauline Antonie,Xie Yuanbin,Sokpor Godwin,Pham Linh,Shomroni Orr,Berulava Tea,Rosenbusch Joachim,Basu Uttiya,Fischer Andre,Nguyen Huu Phuc,Staiger Jochen F,Tuoc Tran Development (Cambridge, England) Fine-tuned gene expression is crucial for neurodevelopment. The gene expression program is tightly controlled at different levels, including RNA decay. N-methyladenosine (m6A) methylation-mediated degradation of RNA is essential for brain development. However, m6A methylation impacts not only RNA stability, but also other RNA metabolism processes. How RNA decay contributes to brain development is largely unknown. Here, we show that Exosc10, a RNA exonuclease subunit of the RNA exosome complex, is indispensable for forebrain development. We report that cortical cells undergo overt apoptosis, culminating in cortical agenesis upon conditional deletion of Exosc10 in mouse cortex. Mechanistically, Exosc10 directly binds and degrades transcripts of the P53 signaling-related genes, such as Aen and Bbc3. Overall, our findings suggest a crucial role for Exosc10 in suppressing the P53 pathway, in which the rapid turnover of the apoptosis effectors Aen and Bbc3 mRNAs is essential for cell survival and normal cortical histogenesis. 10.1242/dev.188276
    N-methyladenosine mediates arsenite-induced human keratinocyte transformation by suppressing p53 activation. Zhao Tianhe,Sun Donglei,Zhao Manyu,Lai Yanhao,Liu Yuan,Zhang Zunzhen Environmental pollution (Barking, Essex : 1987) N-methyladenosine (mA), the most abundant and reversible RNA modification, plays critical a role in tumorigenesis. However, whether mA can regulate p53, a leading antitumor protein remains poorly understood. In this study, we explored the regulatory role of mA on p53 activation using an arsenite-transformed keratinocyte model, the HaCaT-T cell line. We created the cell line by exposing human keratinocyte HaCaT cells to 1 μM arsenite for 5 months. We found that the cells exhibited an increased mA level along with an aberrant expression of the methyltransferases, demethylase, and readers of mA. Moreover, the cells exhibited decreased p53 activity and reduced p53 phosphorylation, acetylation, and transactivation with a high nucleus export rate of p53. Knockdown of the mA methyltransferase, METTL3 significantly decreased mA level, restoring p53 activation and inhibiting cellular transformation phenotypes in the arsenite-transformed cells. Further, using both a bioinformatics analysis and experimental approaches, we demonstrated that mA downregulated the expression of the positive p53 regulator, PRDM2, through the YTHDF2-promoted decay of PRDM2 mRNAs. We showed that mA upregulated the expression of the negative p53 regulator, YY1 and MDM2 through YTHDF1-stimulated translation of YY1 and MDM2 mRNA. Taken together, our study revealed the novel role of mA in mediating arsenite-induced human keratinocyte transformation by suppressing p53 activation. This study further sheds light on the mechanisms of arsenic carcinogenesis via RNA epigenetics. 10.1016/j.envpol.2019.113908
    mA modification-mediated BATF2 acts as a tumor suppressor in gastric cancer through inhibition of ERK signaling. Xie Jian-Wei,Huang Xiao-Bo,Chen Qi-Yue,Ma Yu-Bin,Zhao Ya-Jun,Liu Li-Chao,Wang Jia-Bin,Lin Jian-Xian,Lu Jun,Cao Long-Long,Lin Mi,Tu Ru-Hong,Zheng Chao-Hui,Huang Chang-Ming,Li Ping Molecular cancer BACKGROUND:BATF2, also known as SARI, has been implicated in tumor progression. However, its role, underlying mechanisms, and prognostic significance in human gastric cancer (GC) are elusive. METHODS:We obtained GC tissues and corresponding normal tissues from 8 patients and identified BATF2 as a downregulated gene via RNA-seq. qRT-PCR and western blotting were applied to examine BATF2 levels in normal and GC tissues. The prognostic value of BATF2 was elucidated using tissue microarray and IHC analyses in two independent GC cohorts. The functional roles and mechanistic insights of BATF2 in GC growth and metastasis were evaluated in vitro and in vivo. RESULTS:BATF2 expression was significantly decreased in GC tissues at both the mRNA and protein level. Multivariate Cox regression analysis revealed that BATF2 was an independent prognostic factor and effective predictor in patients with GC. Low BATF2 expression was remarkably associated with peritoneal recurrence after curative gastrectomy. Moreover, elevated BATF2 expression effectively suppressed GC growth and metastasis in vitro and in vivo. Mechanistically, BATF2 binds to p53 and enhances its protein stability, thereby inhibiting the phosphorylation of ERK. Tissue microarray results indicated that the prognostic value of BATF2 was dependent on ERK activity. In addition, the N6-methyladenosine (mA) modification of BATF2 mRNA by METTL3 repressed its expression in GC. CONCLUSIONS:Collectively, our findings indicate the pivotal role of BATF2 in GC and highlight the regulatory function of the METTL3/BATF2/p53/ERK axis in modulating GC progression, which provides potential prognostic and therapeutic targets for GC treatment. 10.1186/s12943-020-01223-4
    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
    RNA demethylase ALKBH5 prevents pancreatic cancer progression by posttranscriptional activation of PER1 in an m6A-YTHDF2-dependent manner. Guo Xingya,Li Kai,Jiang Weiliang,Hu Yangyang,Xiao Wenqin,Huang Yinshi,Feng Yun,Pan Qin,Wan Rong Molecular cancer BACKGROUND:N6-methyladenosine (m6A) is the most abundant reversible methylation modification of eukaryotic mRNA, and it plays vital roles in tumourigenesis. This study aimed to explore the role of the m6A demethylase ALKBH5 in pancreatic cancer (PC). METHODS:The expression of ALKBH5 and its clinicopathological impact were evaluated in PC cohorts. The effects of ALKBH5 on the biological characteristics of PC cells were investigated on the basis of gain-of-function and loss-of-function analyses. Subcutaneous and orthotopic models further uncovered the role of ALKBH5 in tumour growth. mRNA and m6A sequencing and assays of m6A methylated RNA immunoprecipitation-qPCR (MeRIP-qPCR) were performed to identify the targeted effect of ALKBH5 on PER1. P53-binding sites in the ALKBH5 promoter were investigated by ChIP and luciferase assays to reveal the interplay between ALKBH5 and PER1-activated ATM-CHK2-P53/CDC25C signalling. RESULTS:ALKBH5 loss characterized the occurrence and poor clinicopathological manifestations in patients with PC. Overexpression of ALKBH5 reduced tumoural proliferative, migrative, invasive activities in vitro and ameliorated tumour growth in vivo, whereas ALKBH5 knockdown facilitated PC progression. Mechanistically, ALKBH5 posttranscriptionally activated PER1 by m6A demethylation in an m6A-YTHDF2-dependent manner. PER1 upregulation led to the reactivation of ATM-CHK2-P53/CDC25C signalling, which inhibited cell growth. P53-induced activation of ALKBH5 transcription acted as a feedback loop regulating the m6A modifications in PC. CONCLUSION:ALKBH5 serves as a PC suppressor by regulating the posttranscriptional activation of PER1 through m6A abolishment, which may highlight a demethylation-based approach for PC diagnosis and therapy. 10.1186/s12943-020-01158-w
    Fusaric acid decreases p53 expression by altering promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. Ghazi Terisha,Nagiah Savania,Chuturgoon Anil A Epigenetics Fusaric acid (FA) is a food-borne mycotoxin that mediates toxicity with limited information on its epigenetic properties. p53 is a tumour suppressor protein that regulates cell cycle arrest and apoptotic cell death. The expression of p53 is regulated transcriptionally by promoter methylation and post-transcriptionally by N-6-methyladenosine (m6A) RNA methylation. We investigated the effect of FA on p53 expression and its epigenetic regulation via promoter methylation and m6A RNA methylation in human hepatocellular carcinoma (HepG2) cells. HepG2 cells were treated with FA [0, 25, 50, 104, and 150 µg/ml; 24 h] and thereafter, DNA, RNA, and protein was isolated. Promoter methylation and expression of p53 was measured using qPCR and Western blot. RNA immuno-precipitation was used to determine m6A- levels. The expression of m6A methyltransferases ( and ), demethylases ( and ), and readers ( and ) were measured using qPCR. FA induced promoter hypermethylation ( < 0.0001) and decreased expression ( < 0.0001). FA decreased m6A- levels ( < 0.0001) by decreasing ( < 0.0001) and ( < 0.0001); and suppressed expression of ( < 0.0001), ( < 0.0001), and ( < 0.0001) that ultimately reduced p53 translation ( < 0.0001). Taken together, the data shows that FA epigenetically decreased p53 expression by altering its promoter methylation and m6A RNA methylation in HepG2 cells. This study reveals a mechanism for p53 regulation by FA and provides insight into future therapeutic interventions. 10.1080/15592294.2020.1788324
    IGF2BP1 overexpression stabilizes PEG10 mRNA in an m6A-dependent manner and promotes endometrial cancer progression. Zhang Lin,Wan Yicong,Zhang Zihan,Jiang Yi,Gu Zhiyue,Ma Xiaoling,Nie Sipei,Yang Jing,Lang Jinghe,Cheng Wenjun,Zhu Lan Theranostics N6-methyladenosine (mA) mRNA methylation is the most abundant chemical posttranscriptional modification in mRNA and is involved in the regulation of a number of biological processes. Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) has recently been reported as having the capacity to recognize mA sites in mRNA and plays a role in regulating mRNA metabolization. However, it is unclear which genes IGF2BP1 targets to identify mA sites and what are their respective functions in endometrial cancer (EC). Quantitative PCR, western blot and immunohistochemistry were used to measure IGF2BP1 expression in EC cell lines and tissues. Xenograft experiments were performed to examine the role of IGF2BP1 in EC cell growth. RNA-binding protein immunoprecipitation sequencing, methylated RNA-binding protein immunoprecipitation sequencing and RNA-sequencing were also conducted to identify potential IGF2BP1 targets involved in EC regulation. Co-immunoprecipitation and mass spectrometry were used to identify IGF2BP1-interacting proteins. IGF2BP1 expression increased in EC, and high expression of this protein correlated with poor prognosis. IGF2BP1 overexpression/knockdown can promote (and inhibit) cell proliferation and regulate the tumor cell cycle and cancer progression, both and . Mechanistically, IGF2BP1 can recognize mA sites in the 3' untranslated region (3'UTR) of Paternally Expressed Gene 10 (PEG10) mRNA and recruits polyadenylate-binding protein 1 (PABPC1) to enhance PEG10 mRNA stability, which consequently promotes PEG10 protein expression. Additionally, it would appear that a large number of PEG10 proteins bind p16 and p18 gene promoter sequences, thereby repressing expression and accelerating the cell cycle. This investigation found that IGF2BP1 has a crucial role in the mA-dependent regulatory mechanism for endometrial cancer. This study provides new insights into our understanding of disease progression and provides another potential route for understanding biological functions. 10.7150/thno.49345
    Total Panax notoginseng saponin inhibits vascular smooth muscle cell proliferation and migration and intimal hyperplasia by regulating WTAP/p16 signals via mA modulation. Zhu Boqian,Gong Yaoyao,Shen Le,Li Jie,Han Jie,Song Bingzhan,Hu Lianxia,Wang Qingjie,Wang Zhenxing Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie Intimal hyperplasia, the key event of arterial restenosis, is a result of vascular smooth muscle cell (VSMC) proliferation and migration. Previous studies have demonstrated that total Panax notoginseng saponin (TPNS) represses intimal hyperplasia and inhibits the proliferation of VSMCs following balloon injury. However, the underlying roles of TPNS in intimal hyperplasia remain unclear. In this study, we first found that TPNS inhibited the intimal hyperplasia and reversed the reduced mA quantity in balloon catheter-injured rat carotid artery. Then, we measured the expression profiles of mA "writers" (i.e., methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), and WT1 associated protein (WTAP)) and "erasers" (i.e., FTO alpha-ketoglutarate dependent dioxygenase (FTO) and alkB homolog 5, RNA demethylase (ALKBH5)) in vivo and found that TPNS up-regulated the reduced the WTAP expression in balloon catheter-injured rat carotid artery. Furthermore, we illustrated that TPNS inhibited the viability, proliferation, and migration potential of VSMCs via promotion of WTAP expression and suppression of WTAP restored the TPNS-induced inhibition of cell viability, proliferation and migration potential of VSMCs. In addition, we found that p16 was up-regulated in VSMCs treated with TPNS and repression of p16 restored the TPNS-induced inhibition of cell viability, proliferation and migration potential of VSMCs. Finally, we elucidated that, mechanistically, WTAP exerted its role by regulating p16 via mA modification. Collectively, our results reveal the WTAP-p16 signaling axis and highlight the critical roles of m6A modification in intimal hyperplasia. Thus, this study provided a potential biomarker for the assessment of intimal hyperplasia risk following angioplasty as well as a novel therapeutic target for this disease. 10.1016/j.biopha.2020.109935
    m A demethylase ALKBH5 promotes proliferation of esophageal squamous cell carcinoma associated with poor prognosis. Nagaki Yushi,Motoyama Satoru,Yamaguchi Tomokazu,Hoshizaki Midori,Sato Yusuke,Sato Teruki,Koizumi Yukio,Wakita Akiyuki,Kawakita Yuta,Imai Kazuhiro,Nanjo Hiroshi,Watanabe Hiroyuki,Imai Yumiko,Minamiya Yoshihiro,Kuba Keiji Genes to cells : devoted to molecular & cellular mechanisms Esophageal squamous cell carcinoma (ESCC) is one of the most fatal types of malignant tumors worldwide. Epitranscriptome, such as N -methyladenosine (m A) of mRNA, is an abundant post-transcriptional mRNA modification and has been recently implicated to play roles in several cancers, whereas the significance of m A modifications is virtually unknown in ESCC. Analysis of tissue microarray of the tumors in 177 ESCC patients showed that higher expression of m A demethylase ALKBH5 correlated with poor prognosis and that ALKBH5 was an independent prognostic factor of the survival of patients. There was no correlation between the other demethylase FTO and prognosis. siRNA knockdown of ALKBH5 but not FTO significantly suppressed proliferation and migration of human ESCC cells. ALKBH5 knockdown delayed progression of cell cycle and accumulated the cells to G0/G1 phase. Mechanistically, expression of CDKN1A (p21) was significantly up-regulated in ALKBH5-depleted cells, and m A modification and stability of CDKN1A mRNA were increased by ALKBH5 knockdown. Furthermore, depletion of ALKBH5 substantially suppressed tumor growth of ESCC cells subcutaneously transplanted in BALB/c nude mice. Collectively, we identify ALKBH5 as the first m A demethylase that accelerates cell cycle progression and promotes cell proliferation of ESCC cells, which is associated with poor prognosis of ESCC patients. 10.1111/gtc.12792
    WTAP facilitates progression of hepatocellular carcinoma via m6A-HuR-dependent epigenetic silencing of ETS1. Chen Yunhao,Peng Chuanhui,Chen Junru,Chen Diyu,Yang Beng,He Bin,Hu Wendi,Zhang Yanpeng,Liu Hua,Dai Longfei,Xie Haiyang,Zhou Lin,Wu Jian,Zheng Shusen Molecular cancer BACKGROUND:N6-methyladenosine (m6A) methylation, a well-known modification with new epigenetic functions, has been reported to participate in the tumorigenesis of hepatocellular carcinoma (HCC), providing novel insights into the molecular pathogenesis of this disease. However, as the key component of m6A methylation, Wilms tumor 1-associated protein (WTAP) has not been well studied in HCC. Here we investigated the biological role and underlying mechanism of WTAP in liver cancer. METHODS:We determined the expression of WTAP and its correlation with clinicopathological features using tissue microarrays and the Cancer Genome Atlas (TCGA) dataset. And we clarified the effects of WTAP on HCC cells using cell proliferation assay, colony formation, Edu assay and subcutaneous xenograft experiments. We then applied RNA sequencing combined with gene expression omnibus (GEO) data to screen candidate targets of WTAP. Finally, we investigated the regulatory mechanism of WTAP in HCC by m6A dot blot assay, methylated RNA immunoprecipitation (MeRIP) assay, dual luciferase reporter assay, RNA immunoprecipitation (RIP) assay and Chromatin immunoprecipitation (ChIP) assay. RESULTS:We demonstrated that WTAP was highly expressed in HCC which indicated the poor prognosis, and that WTAP expression served as an independent predictor of HCC survival. Functionally, WTAP promoted the proliferation capability and tumor growth of HCC cells in vitro and in vivo. Furthermore, ETS proto-oncogene 1 (ETS1) was identified as the downstream effector of WTAP. The m6A modification regulated by WTAP led to post-transcriptional suppression of ETS1, with the implication of Hu-Antigen R (HuR) as an RNA stabilizer. Then ETS1 was found to inhibit the progression of HCC and could rescue the phenotype induced by WTAP deficiency. Moreover, WTAP modulated the G2/M phase of HCC cells through a p21/p27-dependent pattern mediated by ETS1. CONCLUSION:We have identified that WTAP is significantly up-regulated in HCC and promotes liver cancer development. WTAP-guided m6A modification contributes to the progression of HCC via the HuR-ETS1-p21/p27 axis. Our study is the first to report that WTAP-mediated m6A methylation has a crucial role in HCC oncogenesis, and highlights WTAP as a potential therapeutic target of HCC treatment. 10.1186/s12943-019-1053-8
    Metformin exhibits antiproliferation activity in breast cancer via miR-483-3p/METTL3/mA/p21 pathway. Cheng Lin,Zhang Xu,Huang Yu-Zhou,Zhu Yu-Lan,Xu Ling-Yun,Li Zhi,Dai Xin-Yuan,Shi Liang,Zhou Xu-Jie,Wei Ji-Fu,Ding Qiang Oncogenesis Evidence suggests that metformin might be a potential candidate for breast cancer treatment. Yet, its relevant molecular mechanisms remain to be fully investigated. We found that metformin could suppress the N6-methyladenosine (mA) level in breast cancer cells significantly. The latter has an essential role in breast cancer progression and is newly considered as a therapeutic target. In this study, we measured the mA level by mA colorimetric analysis and dot blot assay. We then performed qRT-PCR, western blot, MeRIP, dual-luciferase reporter assay, and others to explore the mA-dependent pathway associated with metformin. In vivo effect of metformin was investigated using a mouse tumorigenicity model. In addition, breast cancer and normal tissues were used to determine the role of METTL3 in breast cancer. Metformin could reduce the mA level via decreasing METTL3 expression mediated by miR-483-3p in breast cancer. METTL3 is known to be able to promote breast cancer cell proliferation by regulating the p21 expression by an mA-dependent manner. Metformin can take p21 as the main target to inhibit such effect. To specify, this study exhibited that metformin can inhibit breast cancer cell proliferation through the pathway miR-483-3p/METTL3/mA/p21. Our findings suggest that METTL3 may be considered as a potential therapeutic target of metformin for breast cancer. 10.1038/s41389-020-00290-y
    Overexpression of METTL3 attenuates high-glucose induced RPE cell pyroptosis by regulating miR-25-3p/PTEN/Akt signaling cascade through DGCR8. Zha Xu,Xi Xiaoting,Fan Xinyu,Ma Minjun,Zhang Yuanping,Yang Yanni Aging Methyltransferase-like protein 3 (METTL3) regulates multiple cell functions and diseases by modulating N-methyladenosine (mA) modifications. However, it is still unclear whether METTL3 involves in the pathogenesis of diabetic retinopathy (DR). In the present study, we found that high-glucose inhibited RPE cell proliferation, promoted cell apoptosis and pyroptosis in a time-dependent manner. In addition, both METTL3 mRNA and miR-25-3p were low-expressed in the peripheral venous blood samples of diabetes mellitus (DM) patients compared to normal volunteers, and high-glucose inhibited METTL3 and miR-25-3p expressions in RPE cells. As expected, upregulation of METTL3 and miR-25-3p alleviated the cytotoxic effects of high-glucose on RPE cells, and knock-down of METTL3 and miR-25-3p had opposite effects. Additionally, METTL3 overexpression increased miR-25-3p levels in RPE cells in a microprocessor protein DGCR8-dependent manner, and miR-25-3p ablation abrogated the effects of overexpressed METTL3 on cell functions in high-glucose treated RPE cells. Furthermore, PTEN could be negatively regulated by miR-25-3p, and overexpression of METTL3 increased phosphorylated Akt (p-Akt) levels by targeting miR-25-3p/PTEN axis. Consistently, upregulation of PTEN abrogated the protective effects of METTL3 overexpression on RPE cells treated with high-glucose. Collectively, METTL3 rescued cell viability in high-glucose treated RPE cells by targeting miR-25-3p/PTEN/Akt signaling cascade. 10.18632/aging.103130
    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
    Loss of YTHDF2-mediated mA-dependent mRNA clearance facilitates hematopoietic stem cell regeneration. Wang Hu,Zuo Hongna,Liu Jin,Wen Fei,Gao Yawei,Zhu Xudong,Liu Bo,Xiao Feng,Wang Wengong,Huang Gang,Shen Bin,Ju Zhenyu Cell research 10.1038/s41422-018-0082-y
    N6-methyladenosine dynamics in neurodevelopment and aging, and its potential role in Alzheimer's disease. Shafik Andrew M,Zhang Feiran,Guo Zhenxing,Dai Qing,Pajdzik Kinga,Li Yangping,Kang Yunhee,Yao Bing,Wu Hao,He Chuan,Allen Emily G,Duan Ranhui,Jin Peng Genome biology BACKGROUND:N6-methyladenosine (mA) modification is known to impact many aspects of RNA metabolism, including mRNA stability and translation, and is highly prevalent in the brain. RESULTS:We show that mA modification displays temporal and spatial dynamics during neurodevelopment and aging. Genes that are temporally differentially methylated are more prone to have mRNA expression changes and affect many pathways associated with nervous system development. Furthermore, mA shows a distinct tissue-specific methylation profile, which is most pronounced in the hypothalamus. Tissue-specific methylation is associated with an increase in mRNA expression and is associated with tissue-specific developmental processes. During the aging process, we observe significantly more mA sites as age increases, in both mouse and human. We show a high level of overlap between mouse and human; however, humans at both young and old ages consistently show more mA sites compared to mice. Differential mA sites are found to be enriched in alternative untranslated regions of genes that affect aging-related pathways. These mA sites are associated with a strong negative effect on mRNA expression. We also show that many Alzheimer-related transcripts exhibit decreased mA methylation in a mouse model of Alzheimer's disease, which is correlated with reduced protein levels. CONCLUSIONS:Our results suggest that mA exerts a critical function in both early and late brain development in a spatio-temporal fashion. Furthermore, mA controls protein levels of key genes involved in Alzheimer's disease-associated pathways, suggesting that mA plays an important role in aging and neurodegenerative disease. 10.1186/s13059-020-02249-z
    METTL3 plays multiple functions in biological processes. Liu Shuiping,Zhuo Lvjia,Wang Jianjun,Zhang Qin,Li Qiujie,Li Guohua,Yan Lili,Jin Ting,Pan Ting,Sui Xinbing,Lv Qun,Xie Tian American journal of cancer research N-methyladenosine (mA) is the most common internal modification of mRNAs in higher eukaryotic. This process is performed by methyltransferase. Methyltransferase-like 3 (METTL3) is the best known mA methyltransferase that functions in the reversible epi-transcriptome modulation of mA modification. Besides acting as a mA methyltransferase, METTL3 also regulates mRNA translation and other biological processes. In recent years, studies have identified numerous roles and molecular mechanisms associated with METTL3 in multiple biological processes. However, these findings have not been summarized. In this review, we have systematically summarized the most recent important roles of METTL3 in various biological processes, including cell cycle progression, cell proliferation, cell apoptosis, cell migration and invasion, cell differentiation and inflammatory response. In addition, we discuss the prospect of using a METTL3 as a new diagnostic biomarker and therapeutic target for human cancers.
    Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs). Feng Xingmei,Feng Guijuan,Xing Jing,Shen Biyu,Tan Wei,Huang Dan,Lu Xiaohui,Tao Tao,Zhang Jinlong,Li Liren,Gu Zhifeng Cell and tissue research Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-β-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16(INK4A) were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16(INK4A) expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16(INK4A) short interfering RNA. The DNA damage response and p16(INK4A) pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy. 10.1007/s00441-014-1799-7
    METTL3 and ALKBH5 oppositely regulate mA modification of mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes. Song Huiwen,Feng Xing,Zhang Heng,Luo Yunmei,Huang Juan,Lin Meihua,Jin Junfei,Ding Xue,Wu Shujing,Huang He,Yu Tian,Zhang Mukun,Hong Haiou,Yao Shihua,Zhao Yongxiang,Zhang Zhiyong Autophagy N-methyladenosine (mA) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of mA in macroautophagy/autophagy. Here, we show that mA modifications are increased in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mice heart. We found that METTL3 (methyltransferase like 3) is the primary factor involved in aberrant mA modification. Silencing METTL3 enhances autophagic flux and inhibits apoptosis in H/R-treated cardiomyocytes. However, overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 has an opposite effect, suggesting that METTL3 is a negative regulator of autophagy. Mechanistically, METTL3 methylates , a master regulator of lysosomal biogenesis and autophagy genes, at two mA residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with pre-mRNA and subsequently decreases the expression levels of TFEB. Further experiments show that autophagic flux enhanced by METTL3 deficiency is TFEB dependent. In turn, TFEB regulates the expression levels of METTL3 and ALKBH5 in opposite directions: it induces ALKBH5 and inhibits METTL3. TFEB binds to the promoter and activates its transcription. In contrast, inhibition of METTL3 by TFEB does not involve transcriptional repression but rather downregulation of mRNA stability, thereby establishing a negative feedback loop. Together, our work uncovers a critical link between METTL3-ALKBH5 and autophagy, providing insight into the functional importance of the reversible mRNA mA methylation and its modulators in ischemic heart disease. : ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A; CASP3, caspase 3; ELAVL1, ELAV like RNA binding protein 1; FTO, FTO, alpha-ketoglutarate dependent dioxygenase; GFP, green fluorescent protein; GST, glutathione S-transferase; HNRNPD, heterogeneous nuclear ribonucleoprotein D; H/R, hypoxia/reoxygenation; I/R, ischemia/reperfusion; LAD, left anterior descending; mA, N-methyladenosine; MEFs, mouse embryo fibroblasts; Mer, mutated estrogen receptor domains; METTL3, methyltransferase like 3; METTL14, methyltransferase like 14; mRFP, monomeric red fluorescent protein; MTORC1, mechanistic target of rapamycin kinase complex 1; NMVCs, neonatal mouse ventricular cardiomyocytes; PCNA, proliferating cell nuclear antigen; PE, phosphatidylethanolamine; PI, propidium iodide; PTMs, post-translational modifications; PVDF, polyvinylidenedifluoride; RIP, RNA-immunoprecipitation; siRNA, small interfering RNA; SQSTM1, sequestosome 1; TFEB, transcription factor EB; TUBA: tublin alpha; WTAP, WT1 associated protein; YTHDF, YTH N6-methyladenosine RNA binding protein. 10.1080/15548627.2019.1586246
    METTL3-mediated mA modification regulates cell cycle progression of dental pulp stem cells. Luo Haiyun,Liu Wenjing,Zhang Yanli,Yang Yeqing,Jiang Xiao,Wu Shiqing,Shao Longquan Stem cell research & therapy BACKGROUND:Dental pulp stem cells (DPSCs) are a promising cell source in endodontic regeneration and tissue engineering with limited self-renewal and pluripotency capacity. N-methyladenosine (mA) is the most prevalent, reversible internal modification in RNAs associated with stem cell fate determination. In this study, we aim to explore the biological effect of mA methylation in DPSCs. METHODS:mA immunoprecipitation with deep sequencing (mA RIP-seq) demonstrated the features of mA modifications in DPSC transcriptome. Lentiviral vectors were constructed to knockdown or overexpress methyltransferase like 3 (METTL3). Cell morphology, viability, senescence, and apoptosis were analyzed by β-galactosidase, TUNEL staining, and flow cytometry. Bioinformatic analysis combing mA RIP and shMETTL3 RNA-seq functionally enriched overlapped genes and screened target of METTL3. Cell cycle distributions were assayed by flow cytometry, and mA RIP-qPCR was used to confirm METTL3-mediated mA methylation. RESULTS:Here, mA peak distribution, binding area, and motif in DPSCs were first revealed by mA RIP-seq. We also found a relatively high expression level of METTL3 in immature DPSCs with superior regenerative potential and METTL3 knockdown induced cell apoptosis and senescence. A conjoint analysis of mA RIP and RNA sequencing showed METTL3 depletion associated with cell cycle, mitosis, and alteration of METTL3 resulted in cell cycle arrest. Furthermore, the protein interaction network of differentially expressed genes identified Polo-like kinase 1 (PLK1), a critical cycle modulator, as the target of METTL3-mediated mA methylation in DPSCs. CONCLUSIONS:These results revealed mA methylated hallmarks in DPSCs and a regulatory role of METTL3 in cell cycle control. Our study shed light on therapeutic approaches in vital pulp therapy and served new insight into stem cell-based tissue engineering. 10.1186/s13287-021-02223-x
    NSUN2-Mediated m5C Methylation and METTL3/METTL14-Mediated m6A Methylation Cooperatively Enhance p21 Translation. Li Qiu,Li Xiu,Tang Hao,Jiang Bin,Dou Yali,Gorospe Myriam,Wang Wengong Journal of cellular biochemistry N6-methyladenosine (m6A) and m5C methylation are two major types of RNA methylation, but the impact of joint modifications on the same mRNA is unknown. Here, we show that in p21 3'UTR, NSUN2 catalyzes m5C modification and METTL3/METTL14 catalyzes m6A modification. Interestingly, methylation at m6A by METTL3/METTL14 facilitates the methylation of m5C by NSUN2, and vice versa. NSUN2-mediated m5C and METTL3/METTL14-mediated m6A methylation synergistically enhance p21 expression at the translational level, leading to elevated expression of p21 in oxidative stress-induced cellular senescence. Our findings on p21 mRNA methylation and expression reveal that joint m6A and m5C modification of the same RNA may influence each other, coordinately affecting protein expression patterns. J. Cell. Biochem. 118: 2587-2598, 2017. © 2017 Wiley Periodicals, Inc. 10.1002/jcb.25957
    N6-Methyladenosine Induced miR-34a-5p Promotes TNF-α-Induced Nucleus Pulposus Cell Senescence by Targeting SIRT1. Zhu Hao,Sun Bao,Zhu Liang,Zou Guoyou,Shen Qiang Frontiers in cell and developmental biology Low back pain is tightly associated with intervertebral disc degeneration (IVDD) and aberrant nucleus pulposus (NP) is a critical cause. miRNAs N6-methyladenosine (m6A) modification accounts for the TNF-α-induced senescence of NP cells. The aim of this study was to investigate whether m6A modification regulates TNF-α-mediated cell viability, cell cycle arrest, and cell senescence and how it works. The results showed that METTL14 expression positively correlated with m6A and TNF-α expression in HNPCs. The knockdown of METTL14 led to the inhibition of the TNF-α-induced cell senescence. METTL14 overexpression promoted cell senescence. METTL14 regulated the m6A modification of miR-34a-5p and interacted with DGCR8 to process miR-34a-5p. The miR-34a-5p inhibitor inhibited the cell cycle senescence of HNPCs. miR-34a-5p was predicted to interact with the SIRT1 mRNA. SIRT1 overexpression counteracted the miR-34a-5p-promoted cell senescence. METTL14 participates in the TNF-α-induced m6A modification of miR-34a-5p to promote cell senescence in HNPCs and NP cells of IVDD patients. Downregulation of either METTL14 expression or miR-34a-5p leads to the inhibition of cell cycle arrest and senescence. SIRT1 mRNA is an effective binding target of miR-34a-5p, and SIRT1 overexpression mitigates the cell cycle arrest and senescence caused by miR-34a-5p. 10.3389/fcell.2021.642437
    N6-Methyladenosine in RNA and DNA: An Epitranscriptomic and Epigenetic Player Implicated in Determination of Stem Cell Fate. Ji Pengfei,Wang Xia,Xie Nina,Li Yujing Stem cells international Vast emerging evidences are linking the base modifications and determination of stem cell fate such as proliferation and differentiation. Among the base modification markers extensively studied, 5-methylcytosine (5-mC) and its oxidative derivatives (5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-caC)) dynamically occur in DNA and RNA and have been acknowledged as important epigenetic markers involved in regulation of cellular biological processes. N6-Methyladenosine modification in DNA (m6dA), mRNA (m6A), tRNA, and other noncoding RNAs has been defined as another important epigenetic and epitranscriptomic marker in eukaryotes in recent years. The mRNA m6A modification has been characterized biochemically, molecularly, and phenotypically, including elucidation of its methyltransferase complexes (m6A writer), demethylases (m6A eraser), and direct interaction proteins (readers), while limited information on the DNA m6dA is available. The levels and the landscapes of m6A in the epitranscriptomes and epigenomes are precisely and dynamically regulated by the fine-tuned coordination of the writers and erasers in accordance with stages of the growth, development, and reproduction as naturally programmed during the lifespan. Additionally, progress has been made in appreciation of the link between aberrant m6A modification in stem cells and diseases, like cancers and neurodegenerative disorders. These achievements are inspiring scientists to further uncover the epigenetic mechanisms for stem cell development and to dissect pathogenesis of the multiple diseases conferred by development aberration of the stem cells. This review article will highlight the research advances in the role of m6A methylation modifications of DNA and RNA in the regulation of stem cell and genesis of the closely related disorders. Additionally, this article will also address the research directions in the future. 10.1155/2018/3256524
    Epitranscriptomic m6A modification in the stem cell field and its effects on cell death and survival. Chen Jiaxin,Wang Chan,Fei Weiqiang,Fang Xiao,Hu Xiaotong American journal of cancer research The reversible N6-methyl-adenosine (m6A) modification of messenger RNAs (mRNAs) has generated much interest in the field of stem cell modulation in recent years. Meanwhile, mounting evidence has shown that many physiopathological processes concerning cell death and survival harbor this chemical mark. Our review provides an overview of the m6A epitranscriptomic field and the updated mechanisms of m6A decoration in stem cell regulation. Furthermore, we focus on the role of m6A in DNA damage and the immune response, cell apoptosis, autophagy, and senescence, followed by recent advancements in m6A-induced viral replication. The function of abundant RNA-binding proteins (RBPs) identified in m6A regulatory systems will also be discussed in this review, highlighting their far-reaching implications in cellular m6A machinery and disease treatment.
    Decreased expression of mA demethylase FTO in ovarian aging. Sun Xiaoyan,Zhang Yigan,Hu Yuping,An Junxia,Li Lifei,Wang Yiqing,Zhang Xuehong Archives of gynecology and obstetrics PURPOSE:N6-methyladenosine (mA) and demethylase fat mass and obesity-associated protein (FTO) were reported to be associated with oocyte development and maturation. But the relationship between FTO and ovarian aging was still unclear. This study was aimed at investigating the FTO expression level and the mA content during ovarian aging. METHODS:The expression level of FTO and the content of mA RNA methylation in human follicular fluid (FF), granulosa cells (GCs) and mouse ovary from different age groups were studied by ELISA, WB, qRT-PCR, IHC and mA Colorimetric. RESULTS:Human FF ELISA quantified that the level of FTO protein decreased with age (P = 0.025). QRT-PCR results showed that the relative expression of FTO in human GCs was lower in the elderly group than in the young group (P = 0.012). FTO mRNA and protein expression levels were lower in the ovary of 32-week-old mice than in 3- and 8-week-old mice (P < 0.05). Immunohistochemistry showed FTO was relatively decreased in 32-week-old mice (P < 0.05). The mA content in total RNA from old human GCs and ovary from 32-week-old mice was significantly higher compared with the younger ones. CONCLUSIONS:In human FF, GCs and mouse ovary, the expression of FTO decreased while the content of mA increased with aging. However, the inner mechanism still needs further investigation. 10.1007/s00404-020-05895-7
    Profiling of m6A RNA modifications identified an age-associated regulation of AGO2 mRNA stability. Min Kyung-Won,Zealy Richard W,Davila Sylvia,Fomin Mikhail,Cummings James C,Makowsky Daniel,Mcdowell Catherine H,Thigpen Haley,Hafner Markus,Kwon Sang-Ho,Georgescu Constantin,Wren Jonathan D,Yoon Je-Hyun Aging cell Gene expression is dynamically regulated in a variety of mammalian physiologies. During mammalian aging, there are changes that occur in protein expression that are highly controlled by the regulatory steps in transcription, post-transcription, and post-translation. Although there are global profiles of human transcripts during the aging processes available, the mechanism(s) by which transcripts are differentially expressed between young and old cohorts remains unclear. Here, we report on N6-methyladenosine (m6A) RNA modification profiles of human peripheral blood mononuclear cells (PBMCs) from young and old cohorts. An m6A RNA profile identified a decrease in overall RNA methylation during the aging process as well as the predominant modification on proteincoding mRNAs. The m6A-modified transcripts tend to be more highly expressed than nonmodified ones. Among the many methylated mRNAs, those of DROSHA and AGO2 were heavily methylated in young PBMCs which coincided with a decreased steady-state level of AGO2 mRNA in the old PBMC cohort. Similarly, downregulation of AGO2 in proliferating human diploid fibroblasts (HDFs) also correlated with a decrease in AGO2 mRNA modifications and steady-state levels. In addition, the overexpression of RNA methyltransferases stabilized AGO2 mRNA but not DROSHA and DICER1 mRNA in HDFs. Moreover, the abundance of miRNAs also changed in the young and old PBMCs which are possibly due to a correlation with AGO2 expression as observed in AGO2-depleted HDFs. Taken together, we uncovered the role of mRNA methylation on the abundance of AGO2 mRNA resulting in the repression of miRNA expression during the process of human aging. 10.1111/acel.12753
    mA-independent genome-wide METTL3 and METTL14 redistribution drives the senescence-associated secretory phenotype. Liu Pingyu,Li Fuming,Lin Jianhuang,Fukumoto Takeshi,Nacarelli Timothy,Hao Xue,Kossenkov Andrew V,Simon M Celeste,Zhang Rugang Nature cell biology Methyltransferase-like 3 (METTL3) and 14 (METTL14) are core subunits of the methyltransferase complex that catalyses messenger RNA N-methyladenosine (mA) modification. Despite the expanding list of mA-dependent functions of the methyltransferase complex, the mA-independent function of the METTL3 and METTL14 complex remains poorly understood. Here we show that genome-wide redistribution of METTL3 and METTL14 transcriptionally drives the senescence-associated secretory phenotype (SASP) in an mA-independent manner. METTL14 is redistributed to the enhancers, whereas METTL3 is localized to the pre-existing NF-κB sites within the promoters of SASP genes during senescence. METTL3 and METTL14 are necessary for SASP. However, SASP is not regulated by mA mRNA modification. METTL3 and METTL14 are required for both the tumour-promoting and immune-surveillance functions of senescent cells, which are mediated by SASP in vivo in mouse models. In summary, our results report an mA-independent function of the METTL3 and METTL14 complex in transcriptionally promoting SASP during senescence. 10.1038/s41556-021-00656-3