LDHA enhances m6A mRNA methylation through local reduction of 2-ketoglutarate to 2-hydroxyglutarate to suppress FTO activity

N6-methyladenosine (m6A), the most prevalent modification in mRNA, plays critical roles in RNA biology. However, little is known about the regulatory mechanism of RNA m6A in response to stimulus. Here, we show that hypoxia-driven LDHA enhances m6A mRNA methylation through local conversion of 2-ketoglutarate to 2-hydroxyglutarate to suppress FTO activity. Mass spectrometry analysis demonstrated that hypoxia enhanced mRNA m6A modification in multi cell lines. Further knockdown and overexpression screening indicated that hypoxia-driven LDHA enhances mRNA m6A modification through suppression of FTO activity. Moreover, in vitro m6A demethylation assay of FTO showed that LDHA suppressed the demethylation activity of FTO in a NADH-dependent way. And it was found that LDHA facilitated by NADH could catalyze 2-ketoglutarate (2-KG) to L-2-hydroxyglutarate (L-2-HG) to suppress FTO activity. Interestingly, the absolute concentration of LDHA-produced 2-HG was too low to suppress FTO in the in vitro demethylation assay, although 2-HG is indeed a competitive inhibitor of FTO. Further study indicated that LDHA bound FTO, converted 2-KG to 2-HG, resulted in circumambient accumulation of 2-HG to suppress FTO, and consequently enhanced m6A modification. These results shed a light on the regulatory mechanism of RNA m6A modification.