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

Autophagy. 2019 Aug;15(8):1419-1437. doi: 10.1080/15548627.2019.1586246. Epub 2019 Mar 17.

Abstract

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.

Keywords: ALKBH5; HNRNPD; METTL3; cardiomyocytes; hypoxia/reoxyogenation; mA modification.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine / analogs & derivatives*
  • Adenosine / metabolism
  • AlkB Homolog 5, RNA Demethylase / metabolism*
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO / metabolism
  • Animals
  • Apoptosis / drug effects
  • Autophagy / drug effects
  • Base Sequence
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / genetics*
  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors / metabolism
  • Cell Hypoxia / drug effects
  • HEK293 Cells
  • Heterogeneous-Nuclear Ribonucleoprotein D / metabolism
  • Humans
  • Methylation
  • Methyltransferases / metabolism*
  • Mice
  • Myocytes, Cardiac / drug effects
  • Myocytes, Cardiac / metabolism*
  • Oxygen / pharmacology*
  • Promoter Regions, Genetic / genetics
  • RNA Precursors / metabolism
  • RNA Stability / drug effects
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • Rats
  • Transcriptional Activation / genetics
  • Up-Regulation / drug effects

Substances

  • Basic Helix-Loop-Helix Leucine Zipper Transcription Factors
  • Heterogeneous-Nuclear Ribonucleoprotein D
  • RNA Precursors
  • RNA, Messenger
  • Tcfeb protein, mouse
  • N-methyladenosine
  • AlkB Homolog 5, RNA Demethylase
  • FTO protein, mouse
  • Alpha-Ketoglutarate-Dependent Dioxygenase FTO
  • Methyltransferases
  • Mettl3 protein, mouse
  • Adenosine
  • Oxygen

Grant support

This study was supported by grants from National Natural Science Foundation of China (31571241, 31660266), Shanghai Research Project (18ZR1432400), Shanghai University of Medicine&Health Sciences Seed Fund (SFP-18-21-16-001, SFP-18-20-16-006), the key programs of Shanghai Municipal Health and Family Planning Commission (No 201640029), Shanghai Medical Key Specialty Construction Projects (Class A, Series number ZK2015A10) and Zunyi Medical University.