Methylation at the 6 position of adenosine (m6A) in RNA is rapidly and transiently induced at DNA damage sites in response to ultraviolet irradiation. UV light can damage DNA and be a threat to cell survival. The cells respond to this threat by identifying DNA lesions, excising the affected nucleotides and synthesizing new DNA at the site of damage. Yang Shi and colleagues report that polyadenylated RNAs—RNAs to which a tail of adenosines has been added as part of their maturation into functional messenger RNAs—undergo a specific modification when they are close to DNA that has been damaged by UV light. This modification, which consists in the addition of a methyl group to the N6 position of adenosine, is promoted by the METTL3 methyltransferase and suppressed by the FTO demethylase. This RNA modification regulates the DNA repair process, which is delayed in the absence of METTL3. Importantly, the modification helps to recruit the trans-lesion repair DNA polymerase, pol κ, to the damaged DNA site. This study uncovers a new cellular role for this RNA modification. Cell proliferation and survival require the faithful maintenance and propagation of genetic information, which are threatened by the ubiquitous sources of DNA damage present intracellularly and in the external environment. A system of DNA repair, called the DNA damage response, detects and repairs damaged DNA and prevents cell division until the repair is complete. Here we report that methylation at the 6 position of adenosine (m6A) in RNA is rapidly (within 2 min) and transiently induced at DNA damage sites in response to ultraviolet irradiation. This modification occurs on numerous poly(A)+ transcripts and is regulated by the methyltransferase METTL3 (methyltransferase-like 3)1 and the demethylase FTO (fat mass and obesity-associated protein)2. In the absence of METTL3 catalytic activity, cells showed delayed repair of ultraviolet-induced cyclobutane pyrimidine adducts and elevated sensitivity to ultraviolet, demonstrating the importance of m6A in the ultraviolet-responsive DNA damage response. Multiple DNA polymerases are involved in the ultraviolet response, some of which resynthesize DNA after the lesion has been excised by the nucleotide excision repair pathway3, while others participate in trans-lesion synthesis to allow replication past damaged lesions in S phase4. DNA polymerase κ (Pol κ), which has been implicated in both nucleotide excision repair and trans-lesion synthesis5,6, required the catalytic activity of METTL3 for immediate localization to ultraviolet-induced DNA damage sites. Importantly, Pol κ overexpression qualitatively suppressed the cyclobutane pyrimidine removal defect associated with METTL3 loss. Thus, we have uncovered a novel function for RNA m6A modification in the ultraviolet-induced DNA damage response, and our findings collectively support a model in which m6A RNA serves as a beacon for the selective, rapid recruitment of Pol κ to damage sites to facilitate repair and cell survival.

Endothelial nitric oxide synthase (eNOS) plays a central role in maintaining cardiovascular homeostasis by controlling NO bioavailability. The activity of eNOS in vascular endothelial cells (ECs) largely depends on posttranslational modifications, including phosphorylation. Because the activity of AMP-activated protein kinase (AMPK) in ECs can be increased by multiple cardiovascular events, we studied the phosphorylation of eNOS Ser633 by AMPK and examined its functional relevance in the mouse models. Shear stress, atorvastatin, and adiponectin all increased AMPK Thr172 and eNOS Ser633 phosphorylations, which were abolished if AMPK was pharmacologically inhibited or genetically ablated. The constitutively active form of AMPK or an AMPK agonist caused a sustained Ser633 phosphorylation. Expression of gain-/loss-of-function eNOS mutants revealed that Ser633 phosphorylation is important for NO production. The aorta of AMPKα2 ^−/− mice showed attenuated atorvastatin-induced eNOS phosphorylation. Nano–liquid chromatography/tandem mass spectrometry (LC/MS/MS) confirmed that eNOS Ser633 was able to compete with Ser1177 or acetyl-coenzyme A carboxylase Ser79 for AMPKα phosphorylation. Nano-LC/MS/MS confirmed that eNOS purified from AICAR-treated ECs was phosphorylated at both Ser633 and Ser1177. Our results indicate that AMPK phosphorylation of eNOS Ser633 is a functional signaling event for NO bioavailability in ECs.

Significance: The present study identifies a cross-talk of two important posttranslational modifications, revealing that enhancer of zeste homolog 2 (EZH2) O-GlcNAcylation (GlcNac, N -acetylglucosamine) at serine 75 is required for EZH2 protein stability and therefore facilitates the histone H3 trimethylation at K27 to form H3K27me3. The finding is significant because both O-linked GlcNAc transferase-mediated O-GlcNAcylation and EZH2-mediated H3K27me3 formation play a pivotal role in development, and their up-regulation is believed to participate in tumor malignancy. The identification of O-linked GlcNAc transferase association with the polycomb repressive complex 2 (PRC2) further provides a new approach to regulate PRC2 function.