N 6-methyladenosine (m 6 A) mRNA modifications play critical roles in various biological processes. However, no study addresses the role of m 6 A in macroautophagy/autophagy. Here, we show that m 6 A 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 m 6 A 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 m 6 A residues in the 3ʹ-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 m 6 A methylation and its modulators in ischemic heart disease.

In this paper, a new method for analyzing certain physical and chemical properties of liquid water near surfaces is described. "Probe" molecules are dissolved in the water system and are excited by a short laser pulse. The ability of the probe to undergo a fast nonradiative process depends on a reorientational relaxation time of the water solvent, which may become orders of magnitude slower for water near a surface. Using timeresolved methods and a sufficiently fast probe, one can observe a direct dynamic competition between diffusion of the probe and the nonradiative event. Thus, in principle, it is possible to obtain both these rates as a function of distance from a surface. The methods can be applied to a variety of surfaces. Here, they are used to investigate the small biologically relevant water pools in sodium bis(2-ethylhexy1)sulfosuccinate (AOT) reverse micelles, whose surfaces are highly hydrophilic. Perturbations on the translational velocity autocorrelation function of the probe, as measured by the diffusion fluxes, are very large, extending nearly to the center of the largest micelle studied (radius -55 A). On the other hand, perturbations on the orientation relaxation of the solvent, as measured by the probe fluorescence lifetimes, were found to extend no more than -10-15 A from the surface of any of the micelles studied.

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