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. 2019 Apr;15(4):340-347.
doi: 10.1038/s41589-019-0231-8. Epub 2019 Feb 18.

FTO Controls Reversible M 6 Am RNA Methylation During snRNA Biogenesis

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Free PMC article

FTO Controls Reversible M 6 Am RNA Methylation During snRNA Biogenesis

Jan Mauer et al. Nat Chem Biol. .
Free PMC article

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, m1 and m2, reflecting the methylation state of the adenosine adjacent to the snRNA cap. We find that snRNA biogenesis involves the formation of an initial m1 isoform with a single-methylated adenosine (2'-O-methyladenosine, Am), which is then converted to a dimethylated m2 isoform (N6,2'-O-dimethyladenosine, m6Am). The relative m1 and m2 isoform levels are determined by the RNA demethylase FTO, which selectively demethylates the m2 isoform. We show FTO is inhibited by the oncometabolite D-2-hydroxyglutarate, resulting in increased m2-snRNA levels. Furthermore, cells that exhibit high m2-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.

Conflict of interest statement

Competing Interests. S.R.J. is scientific founder, advisor to, and owns equity in Gotham Therapeutics.

Figures

Figure 1 |
Figure 1 |. FTO selectively demethylates small nuclear RNAs.
a, N6-methyladenine (6mA) mapping in total RNA using miCLIP reveals FTO-dependent demethylation of small nuclear RNAs (snRNAs). Shown is the log2 fold-change in methylation of Fto knockout mouse liver (Fto−/−) compared to wild-type liver (WT). Transcripts with significant upregulation (P ≤ 0.05) of transcription-start nucleotide (TSN) methylation are indicated in orange (data represents average from datasets of three independent biological replicates per genotype). b, FTO deficiency leads to increased TSN methylation of major spliceosomal snRNAs. The mean log2 fold-change in TSN methylation of specific snRNA gene classes (U1, U2, U4, U5 and U6) in Fto−/− mouse liver compared to WT is shown (data represents average from datasets of three independent biological replicates per genotype; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; one-way ANOVA with Tukey’s post hoc test ***P≤0.001). c, U1 snRNA shows increased TSN methylation in FTO-deficient mouse liver. Grey tracks denote WT liver, blue tracks denote Fto−/− liver. A representative read coverage track for U1 snRNA is shown (RPM = reads per million mapped reads; data represents combined tracks from datasets of three independent biological replicates per genotype). d, FTO deficiency leads to increased methylation of mature snRNAs. 6mA-immunoblot of WT and FTO−/− HEK293 cells. Methylation of U1, U2, and U6 snRNAs was detected with an antibody directed against 6mA. The left panel shows a representative 6mA-immunoblot with positions of nucleotide size markers and individual snRNAs indicated on the left and right, respectively. The right panel shows the quantification of 6mA signal in U1 and U2 snRNA relative to the 6mA signal in U6 snRNA (n=3 independent biological replicates; mean±s.e.m.; unpaired student’s t-test ***P≤0.001).
Figure 2 |
Figure 2 |. Reversible N6,2’-O-dimethyladenosine (m6Am) in small nuclear RNAs.
a, FTO deficiency reveals the presence of m6Am in small RNAs. Modified adenosines in snRNA caps from wild-type (WT) and FTO-deficient (FTO−/−) HEK293 cells were analyzed by thin-layer chromatography (TLC). The migration position of m6Am and 2’-O-methyladenosine (Am) is indicated by dashed circles. Orange line indicates the small RNA fraction (RNAs<200 nt) used for analysis. Right panel shows quantification of m6Am/Am ratios in small RNA (n=3 independent biological replicates; mean±s.d; unpaired student’s t-test **P≤0.01). b, FTO deficiency reveals the presence of m6Am in U1 snRNA. m6Am and Am in U1 snRNA caps from WT and FTO−/− HEK293 cells were analyzed by TLC. The migration position of m6Am and 2’-O-methyladenosine (Am) is indicated by the dashed black circles. Right panel shows the quantification of the m6Am/Am ratio in the U1-enriched fraction (n=3 independent biological replicates; mean±s.d; unpaired student’s t-test **P≤0.01). c, FTO deficiency reveals the presence of m6Am in U2 snRNA. The relative abundance of m6Am and Am in U2 snRNA caps from WT and FTO−/− HEK293 cells was determined by TLC. (n=3 independent biological replicates; mean±s.d; unpaired student’s t-test ***P≤0.001). d, FTO deficiency increases the relative abundance of m2-snRNA caps. The extended cap structure dinucleotide of small RNA from WT and FTO−/− HEK293 cells was analyzed by LC-MS/MS. Shown is the ratio of m2-snRNA caps (m6Am) to m1-snRNA caps (Am) (n=3 independent biological replicates, mean±s.d.; unpaired Student’s t-test, ***P< 0.001). e, Differential expression of m2-snRNA (cap-ppp-m6Am) caps across cells and tissues. The extended cap structure dinucleotide of small RNA from wild-type HEK293 cells, naïve mouse embryonic stem cells (mESCs), TF-1 erythroleukemia cells, as well as mouse liver and brain tissue was analyzed by LC-MS/MS. Shown is the ratio of m2-snRNA caps to m1-snRNA caps (n.d.=cap-ppp-m6Am not detected; n=3 independent biological replicates, mean±s.d.; unpaired Student’s t-test, ** P<0.01).
Figure 3 |
Figure 3 |. m2-snRNAs are reversibly regulated in oncometabolite-dependent cancer models.
a, Mutant IDH2 increases the abundance of m6Am caps in U1 snRNA. Gel-extracted U1 snRNA from Ctrl and mutant IDH2R140Q-expressing TF-1 cells was analyzed by thin-layer chromatography (TLC). The migration position of m6Am and 2’-O-methyladenosine (Am) is indicated by dashed circles. Orange line indicates the small RNA fraction (RNAs<200 nt) used for analysis. Specific inhibition of mutant IDH2 (AGI-6780) isoforms shows that these effects are reversible by blocking production of 2-HG. Representative images are shown. b, Increased abundance of m6Am caps in U1 snRNA of oncometabolite-dependent cancer cells. Shown is the quantification m6Am/Am ratios in U1 snRNA from Ctrl and mutant IDH2R140Q-expressing TF-1 cells as measured by TLC (n=3 independent biological replicates, mean±s.d.; unpaired Student’s t-test, **P<0.01). c, Increased abundance of m2-snRNA caps in oncometabolite-dependent cancer. The extended cap structure dinucleotide of small RNA from Ctrl, mutant IDH1R132H-, and mutant IDH2R140Q-expressing TF-1 cells was analyzed by LC-MS/MS. Shown is the ratio of m2-snRNA caps (m6Am) to m1-snRNA caps (Am) represented by the integrated peak area ratio of the MRM transition. Blocking production of 2-HG by specific inhibition of mutant IDH1 and IDH2 isoforms with AGI-5027 and AGI-6780, respectively, shows that these effects are reversible (n=3 independent biological replicates, mean±s.d.; one-way ANOVA with Tukey’s post hoc test *P≤0.05, **P≤0.01).
Figure 4 |
Figure 4 |. m2-snRNAs are incorporated into snRNPs.
a, FTO does not exhibit measurable demethylation of m6Am in RNA containing a N2,2,7-trimethylguanosine (TMG) cap. To determine whether FTO demethylates mature snRNA, we incubated full-length human FTO with synthetic 20-mer RNA oligonucleotides starting either with a 5’-m7G-ppp-m6Am or 5’-TMG-ppp-m6Am cap. In the context of an m7G cap, FTO readily converted m6Am to Am (left panels). However, the presence of an TMG cap completely blocked FTO demethylation activity towards m6Am (right panels), indicating that snRNAs starting with a TMG-ppp-m6Am are not a physiological target of FTO (representative HPLC track shown; n=3 independent experiments). b, m2-snRNAs are incorporated into small nuclear ribonucleoproteins (snRNPs). To test whether m2-snRNAs are incorporated into snRNPs, immunoprecipitation of the SmB spliceosomal protein was performed. The relative abundance of modified adenosines in SmB-bound small RNA caps derived from wild-type (WT) and FTO-deficient (FTO−/−) HEK293 cells was determined by thin-layer chromatography. The left panel shows a representative image of the migration pattern of radiolabeled nucleotides, where the position of m6Am and Am is indicated by the dashed black circles. The right panel shows the quantification of the m6Am/Am ratio in small RNA (n=3 independent biological replicates; mean ± s.d; unpaired student’s t-test ***P≤0.001). c, m2-snRNAs have TMG caps. We asked if the presence of m6Am in snRNA blocks the maturation of the snRNA cap from m7G to TMG. The extended cap structure dinucleotide of small RNA from FTO-deficient HEK293 cells and tissues was analyzed by LC-MS/MS. Shown is the ratio of m2-snRNA TMG caps to m1-snRNA. TMG-capped snRNAs were readily detected in HEK293 cells, as well as in mouse liver and brain tissue, demonstrating that, similar to m1-snRNAs, m2-snRNAs are subjected to m7G cap hypermethylation (n=3 independent biological replicates, mean ± s.d.).

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