FTO does not function as an m6A eraser in AML: a reassessment using direct RNA nanopore sequencing

Abstract

A central concept in epitranscriptomics is that the RNA demethylase FTO acts as a site-selective eraser of N6-methyladenosine (m6A) in specific mRNAs. This is most well-established in acute myeloid leukemia (AML), where demethylation of MYC is thought to promote leukemogenesis. Using nanopore direct RNA sequencing to quantify m6A transcriptome-wide, we find that m6A stoichiometry, including at MYC-specific sites, remains unaffected by FTO knockdown or pharmacologic inhibition in AML. Conversely, levels of N6-2'-O-dimethyladenosine (m6Am) are markedly increased in snRNAs, indicating that m6Am, not m6A, is the major substrate of FTO in AML. We extend these results outside the AML context in FTO knockout brain and HEK293T cells, detecting no selective increases in mRNA m6A but widespread increases in snRNA m6Am levels following FTO depletion. These findings suggest that FTO has minimal ability to erase m6A in mRNA. Instead, the reported biological effects of FTO are likely mediated by demethylation of m6Am.