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Autophagy. 2019 Mar 14:1-19. doi: 10.1080/15548627.2019.1586246. [Epub ahead of print]

METTL3 and ALKBH5 oppositely regulate m6A modification of TFEB mRNA, which dictates the fate of hypoxia/reoxygenation-treated cardiomyocytes.

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a Longju Medical Research Center; Key Laboratory of Basic Pharmacology of Ministry of Education , Zunyi Medical University , Zunyi , China.
b Department of Cardiology , Shanghai Chest Hospital, Shanghai Jiao Tong University , Shanghai , China.
c Rutgers Cancer Institute of New Jersey , Rutgers University , New Brunswick , NJ , USA.
d Department of Histology and Embryology, Xiang Ya School of Medicine , Central South University , Changsha , China.
e Research Center of Clinical Pharmacy, State Key Laboratory for Diagnosis and Treatment of Infectious Disease, First Affiliated Hospital , Zhejiang University , Hangzhou , China.
f Laboratory of Hepatobiliary and Pancreatic Surgery , Affiliated Hospital of Guilin Medical University , Guilin , China.
g Department of Cardiology; the First Affiliated Hospital , Harbin Medical University , Harbin , China.
h Department of Cardiology , The ChengGong Hospital Affiliated to Xiamen University , Xiamen , China.
i Digestive Cancer Laboratory , Second Affiliated Hospital of Xinjiang Medical University , Urumqi , China.
j Department of Anesthesia , Affiliated Hospital of Zunyi Medical University , Zunyi , China.
k Health Management Center , The First Affiliated Hospital of USTC (Anhui Provincial Hospital) , Hefei , China.
l Department of Thoracic Surgery, Changhai Hospital , Second Military Medical University , Shanghai , China.
m National Center for International Research of Biological Targeting Diagnosis and Therapy (Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research) , Guangxi Medical University , Nanning , China.
n Department of Surgery; Robert-Wood-Johnson Medical School University Hospital , Rutgers University, State University of New Jersey , New Brunswick , NJ USA.


N6-methyladenosine (m6A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m6A in macroautophagy/autophagy. Here, we show that m6A 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 m6A 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 TFEB, a master regulator of lysosomal biogenesis and autophagy genes, at two m6A residues in the 3'-UTR, which promotes the association of the RNA-binding protein HNRNPD with TFEB 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 ALKBH5 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 m6A methylation and its modulators in ischemic heart disease. Abbreviations: ACTB, actin beta; ALKBH5, alkB homolog 5, RNA demethylase; ANXA5, annexin A5; ATG, autophagy-related; BafA, bafilomycin A1; 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; m6A, N6-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.


ALKBH5; HNRNPD; METTL3; cardiomyocytes; hypoxia/reoxyogenation; mA modification

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