FTO controls reversible m6Am RNA methylation during snRNA biogenesis

Abstract

Small nuclear RNAs (snRNAs) are core spliceosome components and mediate pre-mRNA splicing. Here we show that snRNAs 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 the oncometabolite D-2hydroxyglutarate, 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 controls a previously unknown central step of snRNA processing involving reversible methylation, and suggest that epitranscriptomic information in snRNA may influence mRNA splicing.

Combining Chemical Synthesis and Enzymatic Methylation to Access Short RNAs with Various 5′ Caps

“…Mono‐ or dimethylation of the N 2 ‐position was shown on N 7 Gppp‐RNAs prepared by chemical synthesis and was independent of the sequence or length of the substrate. This allowed access to interesting structural motifs, such as hypermethylated cap RNAs with a 6m A m nucleoside in the first position; thus facilitating research in the methylation and demethylation dynamics of 6m A m in snRNAs . Regarding CMTR1, we found that near‐quantitative 2′‐O‐methylation of the 5′‐terminal nucleotide was achieved on RNA substrates of different lengths, prepared either chemically or enzymatically.…”

“…This modified ribonucleotide is found, in particular, at the transcription‐start nucleotide of Sm‐class spliceosomal snRNAs. The dimethylated m 2 ‐snRNAs are major targets of the RNA demethylase FTO, which removes the methyl group from N 6 ‐adenosine to give m 1 ‐snRNAs …”

“…To determine whether FTO demethylates snRNAs with a monomethylated cap, 7m Gppp 6m A m , or a trimethylated cap, 2m, 2m, 7m Gppp 6m A m , we prepared RNA 7 from 4 (Table ) . Double N 2 ‐methylation was performed on 4 (150 μ m ) with hTgs1 (2.5 μ m ) with SAM at 1 m m , instead of 0.5 m m , as previously used.…”

“…Indeed, after 7.5 h of incubation with hTgs1, trimethylated cap 7 corresponds to the major peak in the HPLC chromatogram, as confirmed by MALDI‐TOF mass analysis (Figure ). Compound 7 was purified by means of RP‐HPLC and 48 nmol of pure material were isolated and used in experiments to investigate FTO demethylation dynamics of snRNAs …”

“…This modification occurs only on 2′‐O‐methylated RNAs, yielding 6m A m , and is theorized to happen co‐transcriptionally upon cap1 formation and to influence mRNA stability . In snRNAs, recent findings suggest that methylation at the N 6 ‐position of the first 2′‐O‐methyl adenosine is dynamic; thus indicating a regulatory function of 6m A m in splicing or other snRNA‐dependent mRNA processing reactions …”