Summary Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. During their biogenesis, snRNAs acquire several constitutive nucleotide modifications. Here we show that snRNAs also contain a regulated and reversible nucleotide modification causing them to exist as two different methyl isoforms, m 1 and m 2 , reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m 1 -isoform with a single-methylated adenosine (2’- O -methyladenosine, Am), which is then converted to a dimethylated m 2 -isoform ( N 6 ,2’- O -dimethyladenosine, m 6 Am). The relative m 1 - and m 2 -isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m 2 -isoform. We show FTO is inhibited by endogenous metabolites, resulting in increased m 2 -snRNA levels. Furthermore, cells that exhibit high m 2 -snRNA levels show altered patterns of alternative splicing. Together, these data reveal that FTO has a central role in snRNA biogenesis and controls a previously unknown step of snRNA processing involving reversible methylation, thereby providing a potential link between reversible RNA modifications and mRNA splicing.

The RNA demethylase FTO targets m6Am in snRNA to establish distinct methyl isoforms that influence splicing