Knockdown Of Fat Mass And Obesity-associated Protein Research Articles

N6-Methyladenosine RNA Demethylase FTO Promotes Gastric Cancer Metastasis by Down-Regulating the m6A Methylation of ITGB1.

Abnormal RNA m6A methylation is known to lead to the occurrence and progression of multiple cancers including gastric cancer (GC). However, the integrative effects of all m6A methylation regulators on GC prognosis are unclear. Our research aimed to globally analyze the prognosis values of all 33 m6A RNA methylation regulators in GC by univariate and multivariate Cox regression analyses. Among all 33 m6A RNA methylation regulators, fat mass and obesity-associated protein (FTO), an m6A demethylase, was identified as a key prognostic risk factor on overall survival (OS) of GC patients. It was found that FTO could promote GC cell migration and invasion abilities, and we predicted that ITGB1 was a demethylated target of FTO. Knockdown (KD) of FTO significantly down-regulated ITGB1 expression at both mRNA and protein levels and augmented ITGB1 mRNA m6A modification level. Moreover, overexpression (OE) of ITGB1 could partially reverse FTO-KD-inhibited migration and invasion of GC cells. Our study found that FTO was an independent risk factor for overall survival (OS) of GC patients and FTO could promote GC metastasis by upregulating the expression of Integrin β1(ITGB1) via decreasing its m6A level. These results indicated that FTO can be a potent GC biomarker for prognosis prediction as well as a potential target in GC treatment.

R-2-HG in AML… friend or foe?

R-2-HG in AML… friend or foe?

R-2-hydroxyglutarate attenuates aerobic glycolysis in leukemia by targeting the FTO/m6A/PFKP/LDHB axis

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)/N6-methyladenosine (m6A)/YTH N6-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 invivo; 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.

Fat mass and obesity-associated protein (FTO) mediates signal transducer and activator of transcription 3 (STAT3)-drived resistance of breast cancer to doxorubicin

ABSTRACT Excessive activation of signal transducer and activator of transcription 3 (STAT3) is implicated in breast cancer (BC) chemoresistance, but its underlying mechanism is not fully understood. There are STAT3 binding sites in fat mass and obesity-associated protein (FTO) promoter region, thus STAT3 may regulate the transcription of FTO. This study aimed to investigate the correlation between FTO and STAT3 in BC chemoresistance. Herein, FTO and STAT3 were highly expressed in doxorubicin-resistant BC (BC-DoxR) cells. CHIP assay verified the binding between STAT3 and FTO promoter in BC-DoxR cells. Dual luciferase reporter assay showed that FTO promoter activity was inhibited by S3I-201 (STAT3 inhibitor) but enhanced by epidermal growth factor (EGF, STAT3 activator) in BC-DoxR and BC cells. FTO mRNA and protein expression were suppressed by S3I-201 in BC-DoxR cells and EGF-stimulated BC cells. Notably, FTO regulated total N6-methyladenosine (m6A) levels in BC-DoxR and BC cells but could not affect STAT3 mRNA expression, indicating that FTO was not involved in the m6A modification of STAT3. However, FTO could activate STAT3 signaling in BC-DoxR and BC cells. Besides, FTO knockdown inhibited the doxorubicin resistance of BC-DoxR cells, while FTO overexpression enhanced the doxorubicin resistance and weakened the doxorubicin sensitivity of BC cells. Moreover, decreased doxorubicin resistance by STAT3 knockdown was abolished by FTO overexpression and decreased doxorubicin sensitivity by STAT3 overexpression was reversed by FTO knockdown, indicating that FTO was implicated in STAT3-mediated doxorubicin resistance and impairment of doxorubicin sensitivity of BC cells. Altogether, our findings provide a mechanism underlying BC doxorubicin resistance.

The N6-methyladenosine modification posttranscriptionally regulates hepatic UGT2B7 expression

UDP-glucuronosyltransferases (UGTs) are enzymes catalyzing the glucuronidation of various endogenous and exogenous compounds. In this study, we examined the possibility that N6-methyladenosine (m6A) modification affects hepatic UGT expression. Treatment of HepaRG cells with 3-deazaadenosine, an inhibitor of RNA methylation, significantly increased UGT1A1, UGT1A3, UGT1A4, UGT1A9, UGT2B7, UGT2B10, and UGT2B15 mRNA levels (1.3- to 2.6-fold). Among them, we focused on UGT2B7 because it most highly contributes to glucuronidation of clinically used drugs. Methylated RNA immunoprecipitation assays revealed that UGT2B7 mRNA in HepaRG cells and human livers is subjected to m6A modification mainly at the 5′ untranslated region (UTR) and secondarily at the 3′UTR. UGT2B7 mRNA and protein levels in Huh-7 cells were significantly increased by double knockdown of methyltransferase-like 3 (METTL3) and METTL14, whereas those were decreased by knockdown of fat mass and obesity-associated protein (FTO) or alkB homolog 5, RNA demethylase (ALKBH5), suggesting that m6A modification downregulates UGT2B7 expression. By experiments using actinomycin D, an inhibitor of transcription, it was demonstrated that ALKBH5-mediated demethylation would attenuate UGT2B7 mRNA degradation, whereas METTL3/METTL14 or FTO-mediated m6A modification would alter the transactivity of UGT2B7. Luciferase assays revealed that the promoter region at −118 to −106 has a key role in the decrease in transactivity of UGT2B7 by FTO knockdown. We found that hepatocyte nuclear factor 4α (HNF4α) expression was significantly decreased by knockdown of FTO, indicating that this would be the underlying mechanism of the decreased transactivity of UGT2B7 by knockdown of FTO. Interestingly, treatment with entacapone, which is used for the treatment of Parkinson’s disease and is an inhibitor of FTO, decreased HNF4α and UGT2B7 expression. In conclusion, this study clarified that RNA methylation posttranscriptionally controls hepatic UGT2B7 expression.

Vascular Smooth Muscle FTO Promotes Aortic Dissecting Aneurysms via m6A Modification of Klf5.

Background: Aortic dissecting aneurysm (ADA) represents an aortic remodeling disease with a high mortality rate. Fat mass and obesity-associated protein (FTO) exerts RNA demethylation function to regulate gene expression related to stem cell differentiation, DNA damage repair, and tumorigenesis, but the role of FTO in ADA is still unclear.Methods: The expression and location of FTO in 43 ADA tissues and 11 normal tissues were determined by RT-qPCR, WB, immunohistochemistry, and immunofluorescence staining. Detecting proliferation and migration of VSMCs. M6A methylated RNA immuno-precipitation qRT-PCR and dual luciferase reporter assay were performed for determining m6A level and interaction between m6A modulation and Klf5 mRNA, respectively.Results: FTO are highly expressed in VSMCs. FTO was positively correlated with BMI, triglyceride, and D-dimer (all P < 0.05). Functionally, both AngII-induced FTO expression and over expression of FTO promote cell proliferation and migration, whereas knockdown of FTO inhibits these functions. Mechanically, we identified Krüppel-like factor 5 (Klf5) as a target of FTO mediating m6A modification. Overexpression of FTO reduced m6A modification on Klf5 mRNA and promoted Klf5 mRNA expression. Furthermore, the p-GSK3β and Klf5 levels increased after FTO overexpression. Finally, knockdown of FTO suppresses the p-GSK3β levels and Klf5 expression regardless of AngII treatment.Conclusions: Our study revealed that FTO expression significantly contributes to the phenotype conversion of VSMCs and the ADA by the demethylation function (m6A), thereby providing a novel therapeutic target.

RNA N6-methyladenosine demethylase FTO regulates PD-L1 expression in colon cancer cells

Fat mass and obesity-associated protein (FTO) is an enzyme that demethylates N6-methyladenosine (m6A), the most abundant RNA modifications in a cell. The upregulated expression of FTO promotes the progression of various types of cancer by modulating cell-intrinsic genes which relate to malignant potential. However, the impact of FTO on the expression of immune-checkpoint molecules in the tumor cells, which are important for immune escape, has not been well understood. We examined the relevance of FTO to programmed cell death-ligand 1 (PD-L1) expression in colon cancer cells. HCT-116 cells showed high expression of both FTO and PD-L1 proteins. The knockdown of FTO by small interfering RNA decreased mRNA and protein levels of PD-L1 in HCT-116 cells. To elucidate the underlying mechanism by which FTO regulates the expression of PD-L1, we depleted FTO in HCT-116 in the presence of IFN-γ, which is a major stimulus to upregulate PD-L1 expression. Depletion of FTO reduced PD-L1 expression in an IFN-γ signaling-independent manner. RNA immunoprecipitation assay revealed the m6A modification of the PD-L1 mRNA and the binding of FTO to the PD-L1 mRNA in HCT-116. Taken together, our results indicated that FTO could regulate PD-L1 expression in colon cancer cells and provides new insights into the regulation of PD-L1 expression by RNA modification.

Fat mass and obesity-associated protein promotes the tumorigenesis and development of liver cancer

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.

DNA Cytosine-Demethylating Agent 5-Aza-2'-Deoxycytidine Targets Leukemia Cells through Reducing DNA N6-Methyladenine

Introduction: It is known that overexpression of DNA methyltransferases (e.g., DNMT1) is frequent and changes of DNA cytosine methylation (5mC) are a constant feature of cancers. DNA methylation inhibitors, such as 5-aza-2'-deoxycytidine (Dec), have been in clinics for patients with leukemia. It is classically believed that promoter hypomethylation coupled by reexpression of epigenetically-suppressed tumor suppressors is a core mechanism behind Dec-impaired leukemia cell growth. However, the fact that global DNA methylation profiling barely predicts Dec-response suggests a demethylation-independent mechanism of Dec-induced cell death. N6-methyladenine (m6A) has been identified recently as an abundant DNA modification in eukaryotes (Wu, Nature 2016;532:329). Importantly, m6A is extensively present in the human genome, and m6A abundance is associated with tumorigenesis (Xie, Cell 2018;71:306). Furthermore, the DNA m6A is dynamically modulated by the methyltransferases (i.e., METTL3, N6AMT1) and demethylases (i.e., ALKBH1), and changes in m6A predict gene expression (Wu, Nature 2016;532:329). Given a potential crosstalk between m6A and distinct epigenetic mechanisms (Yao, Nat. Commun 2017;8:1122), we hypothesized that the anticancer actions of Dec may partially result from changes in DNA m6A in leukemia cells. Methods: Protein expression of target genes was assessed by Western blotting. The levels of DNA cytosine methylation (5mC) and N6-methyladenine (m6A) were measured by dotblotting or liquid chromatography-mass spectrometry (LC-MS/MS). The cell viability and apoptosis were determined by the Cell Counting Kit 8 (CCK8) assays as well as the Annexin V/Propidium Iodide staining and flow cytometry. The peripheral blood mononuclear cells (PBMCs) of leukemia patients from Mayo Clinic were prepared by Ficoll-Hypaque gradient centrifugation. Results: To test our hypothesis, leukemia cells, Kasumi-1, MV4-11, K562 and KU812, were exposed to 2 µM Dec, a clinical achievable concentration, for 72 hours. As expected, Dec treatment led to a downregulation of DNMT1 and DNMT3a, a reduction of 5mC levels by dotblotting using anti-5mC antibody, a blockage of cell proliferation and a promotion of cell apoptosis. When genomic DNA was subjected to dotblotting using anti-m6A antibody, the results revealed a marked decrease of DNA m6A levels in all Dec-treated cells. Then genomic DNA from K562 and MV4-11 cells was enzymatically digested to 2'-deoxynucleosides. dA was quantified by HPLC-UV, while the amount of m6A was measured by isotope dilution HPLC-ESI-MS/MS using 15N labeled internal standard. The standard curves were generated using pure standards, from which the m6A/A ratio was calculated. In agreement with dotblotting results, Dec treatment significantly decreased DNA m6A abundance in both cell lines. Mechanistically, exposure to Dec led to a consistent increase of demethylase fat mass and obesity-associated protein (FTO), but not METTL3 nor ALKBH1 and ALKBH5. Further, knockdown of FTO increased DNA m6A, which was further confirmed by treatment with FTO inhibitors rhein and meclofenamic acid (MA). These data indicate that FTO may be responsible for Dec-induced m6A changes in leukemia cells. To investigate the clinical implications of DNA m6A, we obtained PBMCs from AML patients (n = 10), who received Dec therapy (20 mg/m2 daily for 5 days every 4 weeks) in Mayo Clinic. These PBMCs were further cultured for 48 hours, frozen and stored in 100% ethanol before DNA extraction. The results from dotblotting using anti-5mC or anti-m6A showed that a trend of decrease in both m6A and 5mC abundance is observed, and the pattern of changes in m6A and 5mC displays a positive correlation. Finally, exposure of leukemia cells to the combination of Dec (2 µM) with FTO inhibitor MA (50 µM) induced more cell apoptosis and greater inhibition on cell proliferation as compared to single agent in vitro, supporting FTO inhibitors as new therapeutic agents in leukemia. Conclusion: Our studies suggest that the FTO-DNA m6A axis may partially mediate the therapeutic outcomes of Dec in leukemia. Our findings provide a new mechanistic paradigm for the anticancer activities of Dec, and define the m6A methylation status in leukemia cells as a new pharmacodynamic marker for their response to Dec-based therapy, pointing to a novel treatment strategy for incorporating m6A modulators to enhance the therapeutic index of Dec. Disclosures Al-Kali: Astex Pharmaceuticals, Inc.: Research Funding.

Meclofenamic acid promotes cisplatin-induced acute kidney injury by inhibiting fat mass and obesity-associated protein-mediated m6A abrogation in RNA

The role of RNA methylation on the sixth N atom of adenylate (m6A) in acute kidney injury (AKI) is unknown. FTO (fat mass and obesity-associated protein) reverses the m6A 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 in vitro model, human kidney proximal tubular cells (HK2 cells) were treated with cisplatin. We found that the cisplatin treatment reduces FTO expression and increases m6A levels in vivo and in vitro MA aggravated renal damage and increased apoptosis in cisplatin-treated kidneys, phenotypes that were correlated with reduced FTO expression and increased m6A levels. Moreover, MA promoted apoptosis in cisplatin-treated HK2 cells, which was correlated with the reduced FTO expression and increased m6A in HK2 cells. FTO protein overexpression reduced m6A levels and inhibited apoptosis in cisplatin-treated HK2 cells and also blocked the MA-induced increase in m6A levels and apoptosis rates. In agreement, overexpression of the m6A-generating methyltransferase-like 3 and 14 (METTL3 and METTL14) or siRNA-mediated FTO knockdown promoted apoptosis and enhanced m6A levels in cisplatin-treated HK2 cells. MA increased p53 mRNA and protein levels in AKI both in vitro and in vivo, 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 m6A levels and aggravates renal damages.

Abstract 4022: Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer

Abstract Purpose: N6-methyladenosine (m6A) post-transcriptional RNA modification represents the most abundant epigenetic alteration in eukaryotic cells. These m6A modifications are regulated by a cascade of enzymes and cofactors, categorized as ‘writers’ [methyltransferase-like 3 (METTL3), METTL14, and Wilms’ tumor 1-associating protein (WTAP1)], ‘erasers’ [fat mass and obesity-associated protein (FTO) and alkylated DNA repair protein AlkB homolog 5 (ALKBH5)] and ‘readers’ [YTHDF1, and YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein)]. While emerging evidence indicates that these enzymes play a central role in RNA metabolism (e.g. RNA stability, translation, splicing, transport and localization), their clinical significance, if any, remains unclear. Herein, we for the first time, systematically unraveled the functional role, as well as the clinical significance of m6a regulators in gastric cancer (GC). Experimental design: First, we investigated the expression of the seven m6A regulator genes (FTO, METTL3, METTL14, WTAP, ALKBH5, YTHDF1, and YTHDF2) in a large cohort of 171 GC patients by qRT-PCR assays. Subsequently, a multivariate Cox-regression model was developed using the 7-gene panel to evaluate its predictive potential, followed by Kaplan Meier analyses for survival outcomes. In addition, we undertook a series of functional studies to investigate the oncogenic role of FTO in GC cells, and subsequent validation of our findings in an animal model. Results: Gastric cancer patients with low-expression of METTL3, METTL14, ALKBH5, WTAP and YTHDF1 demonstrated significantly poor OS (p=0.02, 0.001, 0.01, 0.02 and 0.02, respectively), while patients with high FTO expression exhibited markedly worse OS (p&amp;lt;0.0001). Furthermore, the cumulative risk-score derived from these gene panel also significantly associated with poor OS, with a corresponding hazard ratio of 5.47 (95% CI: 3.18-9.41, p&amp;lt;0.0001). We observed that FTO expression was frequently up-regulated in GC cell lines, particularly in those with epithelial-mesenchymal-transition (EMT) features. Functional studies with FTO knockdown in HGC27 and AGS cells inhibited cell proliferation (p=0.005, and &amp;lt;0.0001, respectively) and migratory potential (p&amp;lt;0.0001, respectively); while its overexpression in MKN28 cells resulted in enhanced proliferation and migration (p&amp;lt;0.0001, and 0.007, respectively). Finally, confirming our in-vitro findings, FTO suppression led to significant tumor growth inhibition in a xenograft animal model (p&amp;lt;0.0001). Conclusions: We provide first evidence that m6A regulators may serve as important prognostic biomarkers in GC patients. Our functional studies reveal that FTO is an important oncogene, and may be a novel therapeutic target in patients with gastric cancer. Citation Format: Tadanobu Shimura, Raju Kandimalla, Yuji Toiyama, Yoshinaga Okugawa, Masato Kusunoki, Ajay Goel. Novel evidence for m6A methylation regulators as prognostic biomarkers and potential therapeutic targets in gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4022.

M6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade

Melanoma is one of the most deadly and therapy-resistant cancers. Here we show that N6-methyladenosine (m6A) mRNA demethylation by fat mass and obesity-associated protein (FTO) increases melanoma growth and decreases response to anti-PD-1 blockade immunotherapy. FTO level is increased in human melanoma and enhances melanoma tumorigenesis in mice. FTO is induced by metabolic starvation stress through the autophagy and NF-κB pathway. Knockdown of FTO increases m6A methylation in the critical protumorigenic melanoma cell-intrinsic genes including PD-1 (PDCD1), CXCR4, and SOX10, leading to increased RNA decay through the m6A reader YTHDF2. Knockdown of FTO sensitizes melanoma cells to interferon gamma (IFNγ) and sensitizes melanoma to anti-PD-1 treatment in mice, depending on adaptive immunity. Our findings demonstrate a crucial role of FTO as an m6A demethylase in promoting melanoma tumorigenesis and anti-PD-1 resistance, and suggest that the combination of FTO inhibition with anti-PD-1 blockade may reduce the resistance to immunotherapy in melanoma.

Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.

The biological roles of RNA modifications are still largely not understood. Thus, developing a method for detecting RNA modifications is important for further clarification. We developed a method for detecting RNA modifications called immuno-northern blotting (INB) analysis and herein introduce its various capabilities. This method involves the separation of RNAs using either polyacrylamide or agarose gel electrophoresis, followed by transfer onto a nylon membrane and subsequent immunoblotting using antibodies against modified nucleosides for the detection of specific modifications. We confirmed that INB with the antibodies for 1-methyladenosine (m1A), N6-methyladenosine (m6A), pseudouridine, and 5-methylcytidine (m5C) showed different modifications in a variety of RNAs from various species and organelles. INB with the anti-m5C antibody revealed that the antibody cross-reacted with another modification on DNA, suggesting the application of this method for characterization of the antibody for modified nucleosides. Additionally, using INB with the antibody for m1A, which is a highly specific modification in eukaryotic tRNA, we detected tRNA-derived fragments known as tiRNAs under the cellular stress response, suggesting the application for tracking target RNA containing specific modifications. INB with the anti-m6A antibody confirmed the demethylation of m6A by the specific demethylases fat mass and obesity-associated protein (FTO) and ALKBH5, suggesting its application for quantifying target modifications in separated RNAs. Furthermore, INB demonstrated that the knockdown of FTO and ALKBH5 increased the m6A modification in small RNAs as well as in mRNA. The INB method has high specificity, sensitivity, and quantitative capability, and it can be employed with conventional experimental apparatus. Therefore, this method would be useful for research on RNA modifications and metabolism.

The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation.

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.

MRNA m6A methylation downregulates adipogenesis in porcine adipocytes

Fat Mass and Obesity-associated protein (FTO), associated with obesity, is proved to demethylate N6-methyladenosine (m6A), which raises questions regarding whether m6A plays vital roles in adipogenesis. To prove this, overexpression and knockdown of FTO and METTL3, as well as the chemical treatment in procine adipocytes were conducted. The results showed FTO negatively regulated m6A levels and positively regulated adipogenesis, while METTL3 positively correlated with m6A levels and negatively with adipogenesis. To remove the potential effect of FTO and METTL3 gene, chemical reagents of methylation inhibitor cycloleucine and methyl donor betaine were used to test the regulation effect of m6A on adipogenesis. The results showed the inverse effect of m6A on lipid accumulation in porcine adipocytes. These findings provide compelling evidence that m6A plays a critical role in the regulation of adipogenesis.

N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.

We report here that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of abundant N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA led to an increased level of m6A in mRNA, whereas overexpression of FTO resulted in a decreased level of m6A in human cells. We further show that FTO partially colocalizes with nuclear speckles, supporting m6A in nuclear RNA as a physiological substrate of FTO.