The study reports that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA increases m6A levels in mRNA, while overexpression of FTO decreases m6A levels in human cells. The authors show that FTO partially colocalizes with nuclear speckles, supporting the idea that m6A in nuclear RNA is a physiological substrate of FTO. The study also demonstrates that FTO catalyzes oxidative demethylation of m6A in an iron(II)- and α-KG-dependent manner. In vivo experiments confirm that FTO regulates m6A content in mRNA, with knockdown of FTO leading to an increase in m6A levels and overexpression of FTO resulting in a decrease. Indirect immunofluorescence analysis reveals that FTO partially colocalizes with nuclear speckles, suggesting a dynamic interaction between FTO and nuclear speckles. These findings indicate that m6A in nuclear RNA is a physiological substrate of FTO and that FTO likely affects the processing of pre-mRNA and/or other nuclear RNAs.The study reports that FTO (fat mass and obesity-associated protein) exhibits efficient oxidative demethylation activity of N6-methyladenosine (m6A) residues in RNA in vitro. FTO knockdown with siRNA increases m6A levels in mRNA, while overexpression of FTO decreases m6A levels in human cells. The authors show that FTO partially colocalizes with nuclear speckles, supporting the idea that m6A in nuclear RNA is a physiological substrate of FTO. The study also demonstrates that FTO catalyzes oxidative demethylation of m6A in an iron(II)- and α-KG-dependent manner. In vivo experiments confirm that FTO regulates m6A content in mRNA, with knockdown of FTO leading to an increase in m6A levels and overexpression of FTO resulting in a decrease. Indirect immunofluorescence analysis reveals that FTO partially colocalizes with nuclear speckles, suggesting a dynamic interaction between FTO and nuclear speckles. These findings indicate that m6A in nuclear RNA is a physiological substrate of FTO and that FTO likely affects the processing of pre-mRNA and/or other nuclear RNAs.