Resolving METTL5 Specificity: Direct RNA Sequencing Reveals No Evidence for METTL5 mediated mRNA m6A Methylation in mESCs

However, whether it also methylates mRNAs remains unclear. To address this, we employed direct RNA sequencing (ONT) for m6A detection on native mRNA molecules.

We first validated the quantitative detection of m6A by ONT by treating mESCs with a METTL3 inhibitor. We then compared methylation levels of m6A sites between Mettl5-KO and WT mESCs. Our analysis provides no compelling evidence for METTL5 mediated mRNA methylation in vivo, indicating that its activity is restricted to rRNA.