Increased N-methyladenosine (mA) modifications are observed in H/R-treated cardiomyocytes and ischemia/reperfusion (I/R)-treated mouse hearts.
METTL3 is identified as the primary factor responsible for abnormal mA modifications.
Silencing METTL3 enhances autophagic flux and reduces apoptosis in H/R-treated cardiomyocytes.
Overexpression of METTL3 or inhibition of the RNA demethylase ALKBH5 decreases autophagy.
METTL3 methylates a key regulator of lysosomal biogenesis and autophagy genes, affecting their expression.
, influenced by METTL3 and ALKBH5, plays a crucial role in regulating autophagy and its own expression.
AI simplified
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 . 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 withpre-mRNA and subsequently decreases the expression levels of . 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 thepromoter 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. 6 6 6 6 6 6 6 6 6TFEB TFEB ALKBH5Abbreviations1
Key numbers
10 of 10
Increase in levels
levels were significantly elevated in heart tissues from infarct patients compared to normal tissues.
50%
Decrease in apoptosis
Silencing METTL3 reduced apoptosis rates in H/R-treated NMVCs.
Full Text
We can’t show the full text here under this license. Use the link below to read it at the source.
