Reversible methylation of m6Am in the 5' cap controls mRNA stability

Abstract

Internal bases in mRNA can be subjected to modifications that influence the fate of mRNA in cells. One of the most prevalent modified bases is found at the 5' end of mRNA, at the first encoded nucleotide adjacent to the 7-methylguanosine cap. Here we show that this nucleotide, N⁶,2'-O-dimethyladenosine (m⁶A_m), is a reversible modification that influences cellular mRNA fate. Using a transcriptome-wide map of m⁶A_m we find that m⁶A_m-initiated transcripts are markedly more stable than mRNAs that begin with other nucleotides. We show that the enhanced stability of m⁶A_m-initiated transcripts is due to resistance to the mRNA-decapping enzyme DCP2. Moreover, we find that m⁶A_m is selectively demethylated by fat mass and obesity-associated protein (FTO). FTO preferentially demethylates m⁶A_m rather than N⁶-methyladenosine (m⁶A), and reduces the stability of m⁶A_m mRNAs. Together, these findings show that the methylation status of m⁶A_m in the 5' cap is a dynamic and reversible epitranscriptomic modification that determines mRNA stability.

Extended Data Figure 1. m⁶A enrichment is increased within the 5′ UTR of Fto-knockout mice relative to wild-type mice and FTO prefers m⁶A_m over m⁶A

a, m⁶A peak mass was calculated for all peaks that were found in both Fto-knockout mice (Fto^−/−) and wild-type (WT) mice. The ratio of each individual peak's mass relative to the average peak mass for each sample was then determined, providing a relative peak mass. The relative peak mass for Fto^−/− mice was divided by the relative peak mass for wild-type mice for each m⁶A peak. A metagene analysis was performed to plot the distribution of these peak mass ratios (knockout/wild type) along the length of an mRNA. This analysis reveals that the changes in peak mass ratio for knockout mice relative to wild-type mice are increased in the 5′ UTR. These findings provided the first hint that FTO activity might be directed towards m⁶A_m. The basis for the reduced peak mass ratio in the 3′ UTR is unclear. Because FTO is a demethylating enzyme, loss of FTO should increase nucleotide methylation. Thus, the reduced methylation of m⁶A residues in the 3′ UTR is likely to be an indirect effect of FTO deficiency. CDS, coding sequence; TSS, transcription start site. b, Representative HPLC chromatogram of synthetic standards that were used to determine retention times of adenosine (A), 2′-O-methyladenosin (A_m), N⁶-methyladenosine (m⁶A), or N⁶,2′-O-dimethyladenosine (m⁶A_m). mAU, milli absorbance units. c-f, Reaction curves for FTO with the different substrates that were used to calculate reaction velocity for Michaelis-Menten analysis. FTO concentrations that allowed initial velocity conditions were used for individual oligonucleotides (20 nM FTO for m⁷Gpppm⁶A_m (c) and m⁷Gpppm⁶A (d); 200 nM FTO for m⁷Gpppm⁶A (e) and internal m⁶A (f) in a GGACU context; n = 3 biological replicates; mean ± s.e.m.). g, Michaelis-Menten plots of FTO for either m⁶A_m or m⁶A. Michaelis-Menten curves of FTO reacting with m⁷Gpppm⁶A_m (blue), m⁷Gpppm⁶A (brown), m⁷GpppACm⁶A (orange) or m⁶A in a GGACU context (green). Owing to the increased reaction speed of FTO with m⁶A_m and m⁶A adjacent to the m⁷G compared to more distal m⁶A, the enzyme concentration was tenfold lower when we assessed reaction rates for m⁶A_m (20 nM FTO for the m⁷Gpppm⁶A_m and m⁷Gpppm⁶A oligonucleotide; 200 nM FTO for the m⁷Gpppm⁶A and for internal m⁶A in a GGACU context). Figure 1d shows a plot in which the data are normalized to enzyme concentration. However, here the plot shows data that were not normalized to enzyme concentration (n = 3 biological replicates; mean ± s.e.m.).

Extended Data Figure 2. FTO-mediated demethylation of m⁶A_m depends on integral parts of the mRNA 5′ cap and accurate mass measurement of the oxidative demethylation of the extended m⁷Gpppm⁶A_m-cap by FTO

a, Structure-activity relationship of FTO and its substrate. ALKBH5 preferentially demethylates m⁶A in its physiological sequence context but FTO does not require a sequence context to demethylate m⁶A (refs 3,60). This lack of a sequence preference suggests that m⁶A is not a preferred substrate for FTO. We therefore asked whether FTO preferentially demethylates m⁶A_m in its natural sequence context as the first nucleotide adjacent to the m⁷G cap. To determine the specific structural elements of the extended cap that are required for efficient N⁶-demethylation of m⁶A_m, we synthesized oligonucleotides with different 5′ ends indicated in boxes 1-7. Shown is the amount of product (A_m for substrates 1, 2, 4, 5; A for substrates 3, 6, 7) generated by FTO (200 nM) after 30 min when incubated with different oligonucleotides (20 μM) containing m⁶A_m or N⁶-methyladenosine (m⁶A). The highest FTO demethylation activity on was on the full cap1m structure m⁷Gpppm⁶A_m (1). Removal of the N⁷-methyl from the guanosine (2) reduced FTO activity by 30% (2), whereas removal of either the 2′-O-methyl from the adenosine (3) or the m⁷G (4) resulted in a 50% activity loss. FTO activity was further reduced by removal of m⁷Gpp (5). The lowest FTO demethylation activity was observed when using m⁶A as a substrate, either at the +3 position after the cap (6) or internally in a GGACU context (7). Thus, an adjacent m⁷G cap does not activate m⁶A as a substrate for FTO. These results indicate that FTO activity is dependent on the presence of a full cap structure, including the 2′-O-methyl at the +1 position, whereas m⁶A is a poor substrate for FTO (one-way ANOVA with Tukey's post hoc test; *P< 0.001; n = 3 biological replicates; mean ± s.e.m.). b, Structure-activity relationship of FTO and its substrate. Shown is the amount of substrate converted by FTO in a time-dependent manner at the same reaction conditions as in a (two-way ANOVA with Tukey's post hoc test; *P < 0.001 versus all other structures; n = 3 biological replicates; mean ± s.e.m.). c, d, FTO demethylates m⁷Gpppm⁶A_m at the N⁶-position through oxidization of m⁷Gpppm⁶A_m to an N⁶-hydroxymethyl intermediate (m⁷Gppphm⁶A_m). The final reaction product is m⁷GpppA_m. Liquid chromatography/mass spectrometry analysis of m⁷Gpppm⁶A_m RNA either left untreated (c; —FTO) or after incubation with 3 μM FTO for 10 min (d; +FTO). Shown are representative mass-to-charge (m/z) ratios of precursor ions. In the absence of FTO, the dinucleotide shows a measured m/z ratio of 813.1173, 0.98 p.p.m. mass accuracy from the exact m/z of 813.1165 (formula C₂₃H₃₃N₁₀O₁₇P₃). Incubation with FTO generates m⁷Gppphm⁶A_m, shown as a measured m/z of 829.1123, 1.01 p.p.m. mass accuracy from the exact m⁷Gppphm⁶A_m m/z of 829.1114 (formula C₂₃H₃₃N₁₀O₁₈P₃). The demethylated final product m⁷GpppA_m and residual non-demethylated m⁷Gpppm⁶A_m were also detected in the FTO reaction mixture, with m⁷GpppA_m showing a measured m/z of 799.1064, 6.9 p.p.m. mass accuracy from the exact m/z of 799.1009 (formula C₂₂H₃₁N₁₀O₁₇P₃).

Extended Data Figure 3. ⁶A_m is the preferred substrate for FTO in vivo

a, Modifications of the extended mRNA cap. The first nucleotide adjacent to the m⁷G and the 5′-to-5′-triphosphate (ppp) linker is subjected to 2′-O-methylation (orange) on the ribose, forming cap1. Cap1 can be further 2′-O-methylated at the second nucleotide to form cap2 (not depicted here). If cap1 contains a 2′-O-methyladenosine (A_m), it can be further converted to cap1m by N⁶-methylation (blue), which results in N⁶,2′-O-dimethyladenosine (m⁶A_m). b, Relative abundance of m⁶A in mRNA treated with recombinant FTO. Internal m⁶A residues that follow G in mRNA can be labelled and quantified in a 2D TLC method. The relative abundance of m⁶A versus (A + C + U) in 400 ng mRNA that was either left untreated (—FTO) or incubated for 1 h with 1 μM bacterially expressed recombinant human FTO (+FTO) was determined by 2D TLC. We did not observe any decrease of m⁶A in FTO-treated mRNA, indicating that FTO does not efficiently demethylate m⁶A in its physiological context in mRNA in vitro (representative images shown; n = 3 biological replicates; mean ± s.e.m.). c, FTO with a nuclear export signal is localized in the cytoplasm. Immunofluorescence staining of DDDDK/Flag tag in HEK293T cells transfected with Flag-tagged wild type FTO (Flag-FTO) or Flag-tagged FTO with an N-terminal nuclear export signal (NES-FTO). FTO is primarily nuclear while NES-FTO is readily detected in the cytosol. DAPI was used to stain nuclei (representative images shown). d, Western blot analyses were performed to verify successful knockdown, overexpression and knockout. Top left, cell extracts from HEK293T cells with FTO knockdown were blotted with anti-FTO antibody. Knockdown efficiency was approximately 75%. The cell extracts were from the same samples used for RNA-seq analysis in Fig. 3d. GAPDH was used as loading control. Top right, western blot analysis of HEK293T expressing Flag vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) that were used for RNA-seq half-life analysis in Fig. 3c. An antibody directed against β-actin was used as a loading control. The lower band represents endogenous FTO, whereas the upper band represents exogenous NES-FTO, which showed approximately tenfold overexpression. Top left, cell extracts from ALKBH5-knockdown HEK293T cells with were blotted with anti-ALKBH5 antibody. Knockdown efficiency was approximately 90%. The cell extracts were from the same samples used for RNA-seq analysis in Extended Data Fig. 7e. β-Actin was used as loading control. Top right, western blot analysis of three different HEK293T clonal lines with CRISPR-mediated knockout of DCP2 that were used for RNA-seq analysis. GAPDH was used as a loading control. e, FTO expression decreases m⁶A_m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T expressing Flag vector (Ctrl) or Flag-tagged FTO with an N-terminal nuclear export signal (Flag-NES-FTO) was determined by 2D TLC. When determining the ratio of m⁶A_m to A_m, we observed a significant decrease of m⁶A_m in Flag-NES-FTO-overexpressing cells, indicating that FTO can convert cytoplasmic m⁶A_m to A_m in vivo. Notably, the ratios of m⁶A_m:A_m that we observed upon FTO expression (both with and without the NES) may under-represent the true effect of FTO: A_m mRNAs are generally less stable than m⁶A_m mRNAs owing to their degradation in cells via DCP2-mediated pathways (see Figs 3 and 4). Thus the A_m mRNAs generated by FTO-mediated demethylation of m⁶A_m may not efficiently accumulate in cells compared to m⁶A_m mRNAs (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P ≤ 0.01). f, FTO expression does not affect m⁶A in HEK293T cells. The relative abundance of m⁶A versus (A + C + U) in mRNA of HEK293T expressing empty vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) was determined by 2D TLC. We did not observe any decrease of m⁶A upon NES-FTO expression, indicating that FTO does not readily influence levels of m⁶A in HEK293T cells at this level of expression. Notably, under these same expression conditions, m⁶A_m is readily demethylated (see Extended Data Fig. 3e) (representative images shown; n = 3 biological replicates; mean ± s.e.m.). Control experiments measuring m⁶A and m⁶A_m levels following ALKBH5-knockdown and expression in HEK293T cells are shown in Extended Data Fig. 4. g, FTO deficiency increases m⁶A_m in vivo. Relative abundance of modified adenosines in mRNA caps of embryonic day (E) 14 wild-type (WT) littermate controls and Fto knockout (Fto^–/–) mouse embryos (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, **P≤ 0.01). h, FTO knockdown does not affect m⁶A in HEK293T cells. The relative abundance of m⁶A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against FTO (siFTO) was determined by 2D TLC. We did not observe any increase of m⁶A upon FTO knockdown, indicating that FTO does not readily influence levels of m⁶A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.). i, Relative abundance of m⁶A in Fto-knockout mouse embryos. The relative abundance of m⁶A versus (A + C + U) in mRNA of embryonic day 14 wild-type littermate controls and Fto-knockout (Fto^−/−) mouse embryos was determined by 2D TLC. We did not observe any increase of m⁶A in Fto-deficient embryos, indicating that FTO does not influence the levels of m⁶A in this embryonic stage (representative images shown; n = 3 biological replicates; mean ± s.e.m.).

Extended Data Figure 4. ALKBH5 demethylates m⁶A but not m⁶A_m in mRNA in HEK293T cells

a, ALKBH5 expression does not decrease m⁶A_m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST–ALKBH5) was determined by 2D TLC. When determining the ratio of m⁶A_m to A_m, we did not observe a significant decrease of m⁶A_m in ALKBH5-overexpressing cells, indicating that ALKBH5 does not convert m⁶A_m to A_m in vivo (representative images show n; n = 3 biologic al replic ates; me an ± s.e.m.). b, ALKBH5 knockdown does not increase m⁶A_m in HEK293T cells. The relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. When determining the ratio of m⁶A_m to A_m, we did not observe a significant increase of m⁶A_m in ALKBH5-expressing cells, indicating that ALKBH5 does not convert m⁶A_m to A_m in vivo (repres entative images shown; n = 3 biological replicates; mean ± s.e.m.). c, ALKBH5 knockdown increases m⁶A in HEK293T cells. The relative abundance of m⁶A versus (A + C + U) in mRNA of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against ALKBH5 (siALKBH5) was determined by 2D TLC. We observed an approximately 30% increase of m⁶A upon ALKBH5 knockdown, indicating that ALKBH5 readily influences the levels of m⁶A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P ≤ 0.05). d, ALKBH5 expression decreases m⁶A in HEK293T cells. The relative abundance of m⁶A versus (A + C + U) in mRNA of HEK293T cells expressing GST vector (Ctrl) or ALKBH5 with an N-terminal GST tag (GST-ALKBH5) was determined by 2D TLC. We observed a significant decrease of m⁶A upon ALKBH5 expression, indicating that SLKBH5 readily influences levels of m⁶A in vivo (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, **P ≤ 0.01).

Extended Data Figure 5. Newly mapped m⁶A_m clusters overlap with transcription start sites (TSS) and the YYANW initiator core motif and mark mRNAs for increased half-life

a, b, To confirm that that the residues identified as m⁶A_m in miCLIP reflect transcription initiation sites, we searched for known TSS and transcription initiation sequences around each m⁶A_m-containing region. Notably, owing to the calling algorithm, these regions do not contain any 5′ UTR m⁶A. To identify genome-wide positions of the TSS we used published CAGE-seq datasets (see Methods). Shown is the nucleotide distance of the called m⁶A_m from TSS (a) and YYANW (b). These results demonstrate that TSS and the YYANW core initiator sequence are highly clustered at m⁶A_m-containing regions (5′-most nucleotide is at position 0 on the x-axis). This suggests that the called m⁶A_m-containing regions reflect true TSS. c, Related to Fig. 3a, c. Correlation of half-life replicates derived from Flag-transfected (Ctrl, left scatter plot) or Flag-NES-FTO-transfected (NES-FTO, right scatter plot) HEK293T cells. The Pearson correlation coefficient (r) is shown for each comparison and indicates high correlation between replicates. d, mRNA stability is determined by the modification state of the first encoded nucleotide in HeLa cells. Cumulative distribution plot of the half-life for mRNAs that start with m⁶A_m, A_m, C_m, G_m and U_m. The half-life of mRNAs starting with an m⁶A_m is approximately 2.5 h longer compared to mRNAs starting with A_m, C_m, G_m or U_m. Notably, for this analysis we used m⁶A_m mRNAs identified in HEK293T cells to analyse published half-life data sets from HeLa cells. This allowed us to determine if the stabilizing effect of m⁶A_m on mRNA half-lives is conserved across different cell types. Indeed, the increase in m⁶A_m mRNA half-life compared to other starting nucleotides was similar to what we observed in Fig. 3a (n = 2,401 (m⁶A_m); 645 (A_m); 1,310 (C_m); 988 (G_m); 1,533 (U_m); data represents the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ***P<2.2 × 10⁻¹⁶ versus m⁶A_m). e, related to Extended Data Fig. 6d, g. Correlation of half-life replicates derived from published HeLa cell datasets. The Pearson correlation coefficient (r) is shown and indicates high correlation between replicates. f, Stable mRNAs show enrichment of m⁶A_m miCLIP reads in HEK293T cells. miCLIP involves recovery of RNA fragments that interact with a 6mA-specific antibody, and thus recover m⁶A- and m⁶A_m-containing RNA fragments. The sequenced fragments, or miCLIP reads, map internally when they are m⁶A. However, m⁶A_m maps at the 5′ ends of transcripts. To determine whether mRNAs with long half-life show m⁶A_m enrichment, we performed metagene analysis of HEK293T cell-derived miCLIP tag distribution in mRNAs that are in the top quartile of mRNA stability (blue) and the bottom quartile of mRNA stability (orange). The miCLIP tag distribution of all mRNAs is shown as a grey dashed line. On all mRNAs, miCLIP reads were enriched around the stop codon, a pattern that reflects the typical distribution of m⁶A in mRNA. Additional enrichment of miCLIP reads was seen in the 5′ UTR, which we previously showed primarily reflects m⁶A_m residues. However, when we examined mRNAs with a long half-life (≥ 10 h), we observed a pronounced enrichment of miCLIP reads in the 5′ UTR. In contrast, mRNAs with a short half-life (≤3 h) exhibit markedly fewer miCLIP reads in the 5′ UTR. These data suggest that m⁶A_m is associated with increased mRNA stability (n = 10,123 (all mRNAs); 820 (short half-life); 2,871 (long half-life)). g, Stable mRNAs show enrichment of m⁶A_m miCLIP reads in HeLa cells. Similar to f, however, for this analysis we used miCLIP reads derived from HEK293T cells to analyse published half-life datasets from HeLa cells. A marked enrichment of miCLIP reads was seen in the 5′ UTR of stable mRNAs, indicating elevated prevalence of m⁶A_m in these mRNAs. These data suggest that m⁶A_m is associated with increased mRNA stability, not only in HEK293T cells but also in HeLa cells. Importantly, the results are quantitatively similar to the results shown in f, indicating that m⁶A_m mRNAs identified in HEK293T cells behave similarly in HeLa cells. (n = 18,286 (all mRNAs); 4,552 (short half-life); 3,619 (long half-life)).

Extended Data Figure 6. m⁶A_m mRNAs show increased translation efficiency

a, mRNA translation efficiency is associated with the modification state of the first encoded nucleotide in HEK293 cells. Cumulative distribution plot of the translation efficiency for mRNAs that start with m⁶A_m, A_m, C_m, G_m and U_m. The translation efficiency of mRNAs starting with an m⁶A_m is significantly higher compared to mRNAs starting wit h A_m, C_m, G_m or U_m (n = 3,024 (m⁶A_m); 921 (A_m); 1,788 (C_m); 1,351 (G_m); 2,008 (U_m); data represent the average from two independent previously published ribosome profiling data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, *P≤2.3 × 10⁻² versus m⁶A_m). b, Correlation of translation efficiency replicates derived HEK293T cells. The Pearson correlation coefficient (r) is shown. c, Distribution of reads between the coding sequence (CDS) and UTRs. High coverage in the CDS compared to UTRs verifies ribosome-derived footprints. d, Total number of ribosome footprints near the start and stop codon of transcripts. e, Three-nucleotide periodicity demonstrates ribosome-derived footprints. f, Position of ribosome footprints relative to the reading frame.

Extended Data Figure 7. Expression changes of m⁶A_m, m⁶A and A_m mRNAs upon NES-FTO expression and FTO or ALKBH5 deficiency

a, m⁶A_m mRNAs exhibit increased half-life compared to A_m mRNAs in vivo.HEK293T cells were electroporated with in vitro-synthesized mRNAsstarting with either of two extended caps: m⁷Gppp A_m or m⁷Gpppm⁶A_m.We then isolated cellular poly(A) RNA and determined the in vivo half-life of the electroporated A_m- and m⁶A_m-containing mRNA by qRT-PCR.In control siRNA-treated HEK293T cells (siCtrl), the m⁶A_m mRNAshowed a trend towards increased half-life compared to the A_m mRNA(unpaired Student's t-test, P = 0.08). Notably, when we performed the sameexperiment in FTO siRNA-treated cells (siFTO) to prevent demethylationof m⁶A_m, the m⁶A_m mRNA half-life was significantly increased (n = 3biological replicates; mean ± s.e.m.; unpaired Student's t-test, P ≤ 0.05). b, NES-FTO expression preferentially affects the half-life of m⁶A_m mRNAscompared to m⁶A mRNAs. Changes in half-life of mRNAs containingeither m⁶A_m or m⁶A in HEK293T cells transfected with either Flag vector(Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO)were determined by RNA-seq. m⁶A_m mRNAs are generally long-lived (see Fig. 3a) and show reduced half-lives after NES–FTO expression. We asked if FTO could elicit a similar effect on mRNAs containing m⁶A. For this experiment, we used a set of mRNAs with annotated m⁶A residues, excluding those which also contain an annotated m⁶A_m. NES-FTO expression reduced the half-life of m⁶A_m mRNAs but did not have any substantial effect on the half-life of m⁶A mRNAs. These data support the idea that FTO preferentially targets m⁶A_m compared to m⁶A (n = 2,049 (m⁶A_m); 2,495 (m⁶A); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ***P ≤ 2.2 × 10⁻¹⁶ versus m⁶A). c, NES-FTO expression preferentially affects the half-life of m⁶A_m mRNAs compared to A_m mRNAs. Changes in half-life of A_m mRNAs (FUCA1, PCK1, SCFD2) and m⁶A_m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. m⁶A_m mRNAs show a significant reduction in half-life after NES-FTO expression whereas the half-life of A_m mRNAs is less affected. These data examine specific mRNAs in contrast to the whole-transcriptome analysis presented in Fig. 3c and also demonstrate the stabilization effect of m⁶A_m using a different method to measure mRNA half-life (that is, BrU pulse-chase labelling) other than transcriptional inhibition (n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P ≤ 0.05, **P ≤ 0.01). d, The expression of mRNAs containing either m⁶A_m or A_m upon Fto knockout was determined by RNA-seq. FTO depletion (Fto^−/−) results in increased abundance of mRNAs with an annotated m⁶A_m residue in liver tissue derived from Fto-knockout mice. Fold change was measured relative to the RNA levels measured in the same tissue obtained from wild-type littermates (n = 2,048 (m⁶A_m); 1,025 (A_m); 2,081 (C_m); 1,742 (G_m); 1,242 (U_m); data represent the average from two independent data sets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ***P ≤ 7.5 × 10⁻⁶ m⁶A_m versus A_m and U_m). e, Knockdown of ALKBH5 does not increase the levels of m⁶A_m mRNAs. The expression of mRNAs containing either m⁶A_m or A_m upon ALKBH5 knockdown in HEK293T cells was determined by RNA-seq. In contrast to knockdown or knockout of FTO, m⁶A_m mRNAs are slightly less abundant than A_m mRNAs in ALKBH5-knockdown cells. This suggests that ALKBH5 does not target m⁶A_m-containing mRNAs in vivo (n = 3,111 (m⁶A_m); 1,928 (A_m); 4,382 (C_m); 3,110 (G_m); 3,998 (U_m); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, **P ≤ 1.2 × 10⁻³ m⁶A_m versus A_m and U_m).

Extended Data Figure 8. m⁶A_m mRNAs are resistant to DCP2-mediated decapping and microRNA-mediated gene silencing

a, DCP2 decapping products are m⁷GDP. Here we confirm the identity of the putative m⁷GDP decapping product in the decapping assay by treatment with nucleoside-diphosphate kinase (NDPK). The shift to the m⁷GTP position confirms that the released product is m⁷GDP. A cap-labelled RNA with a guanosine as the first nucleotide was used as a positive control (lanes 3, 6, 9; the red ‘p’ denotes the position of the ³²P). b, Michaelis-Menten curves of 10 nM DCP2 reacting with m⁷Gpppm⁶A_m (blue) or m⁷GpppA_m (orange) for 30 min at 37 °C. DCP2 shows higher decapping activity towards m⁷GpppA_m than to m⁷Gpppm⁶A_m (the dashed lines indicate the K_m on the × axis; n = 3 biological replicates; mean ± s.e.m.). c, DCP2 depletion preferentially stabilizes A_m mRNAs compared to m⁶A_m mRNAs. Changes in half-life of A_m mRNAs (FUCA1, PCK1, SCFD2) and m⁶A_m mRNAs (PCNA, PSMD3, MAGOHB) in HEK293T cells transfected with either Flag vector (Ctrl) or DCP2-knockout cells (DCP2^−/−) were determined by BrU pulse-chase analysis and subsequent qRT-PCR. A_m mRNAs show a significant increase in half-life after DCP2 depletion whereas the half-life of m⁶A_m mRNAs was not significantly increased. These data are related to the whole-transcriptome expression analysis presented in Fig. 4c and indicate that, in addition to the observed abundance changes of non-m⁶A_m mRNAs versus m⁶A_m mRNAs, DCP2 also selectively affects the half-life of specifically examined mRNAs (n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P ≤ 0.05, **P ≤ 0.01). d, In Fig. 4d, we found that m⁶A_m mRNAs show less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to further examine this concept using additional independent datasets of gene expression following depletion of proteins required for microRNA-mediated mRNA degradation, such as members of the Argonaute protein family. Measurement of mRNA expression in AGO2-knockdown HEK293T cells (siAGO2) compared to control cells (siCtrl) revealed more pronounced upregulation of non-m⁶A_m mRNAs compared to those that have m⁶A_m (n = 2,080 (m⁶A_m); 596 (A_m); 1,085 (C_m); 805 (G_m); 1,274 (U_m); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ***P ≤ 1 × 10⁻⁴ m⁶A_m versus A_m, C_m and U_m). e, Similar to Fig. 4d, but here we only look at the expression changes of mRNAs that contain TargetScan-predicted microRNA-binding sites. Applying this filter criteria, we also observed that DICER knockdown in HEK293T cells (siDICER) resulted in more pronounced upregulation of non-m⁶A_m miRNA target mRNAs compared to those that have m⁶A_m (n = 1,208 (m⁶A_m); 359 (A_m); 607 (C_m); 467 (G_m); 713 (U_m); data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, ***P ≤ 9.6 × 10⁻⁴ versus m⁶A_mN_m, where N_m = A_m, C_m, G_m or U_m). f, In Fig. 4d and Extended Data Fig. 8e we show that m⁶A_m mRNAs exhibit less upregulation upon DICER knockdown than mRNAs beginning with other nucleotides. We wanted to examine this concept further using additional filtering criteria. Thus, we asked if m⁶A_m mRNA resistance to DICER depletion is dependent on the number of microRNA-binding sites. Therefore, we divided mRNAs into five groups: mRNAs that do not contain a predicted microRNA-binding site (0) and mRNAs that belong to specific quartiles that we assigned depending on the number of microRNA-binding sites (low (1) to high (4)). Notably, we did not observe any expression difference between m⁶A_m mRNAs and non-m⁶A_m mRNAs that do not carry predicted microRNA-binding sites. However, there was a clear increase in mRNA expression for mRNAs that contain microRNA-binding sites, and this increase was dependent on the number of microRNA-binding sites. Notably, for each quartile, m⁶A_m mRNAs were significantly less upregulated than N_m mRNAs (n = 91 versus 89 (m⁶A_m versus N_m; 1), 252 versus 339 (m⁶A_m versus N_m; 1), 311 versus 454 (m⁶A_m versus N_m; 2), 247 versus 541 (m⁶A_m versus N_m; 3), 229 versus 512 (m⁶A_m versus N_m; 4); data represent the average from two independent datasets; number of microRNA-binding sites in each quartile: 1 = 1–3; 2 = 4-6; 3 = 7–12; 4 = 13-54; 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 ≤ 0.05, ***P ≤ 0.001, n.s., not significant). g, In Fig. 4d and Extended Data Fig. 9d-f we show that m⁶A_m mRNAs are largely resistant to expression changes upon global inhibition of the microRNA machinery. We next asked whether introduction of a single microRNA also leads to differential responses of m⁶A_m mRNAs compared to non-m⁶A_m mRNAs. We used a published dataset where HeLa cells were transfected with a miR-155 duplex to achieve microRNA-specific mRNA degradation. For this analysis, we used m⁶A_m mRNAs mapped in HEK293T cells. We first asked if we could observe a differential effect of mRNA degradation on miR-155 target and non-target mRNAs in the HeLa cell dataset. Indeed, miR-155 target mRNAs were significantly more suppressed in miR-155-transfected HeLa cells. This confirms that miR-155 target mRNA degradation can be detected in this dataset (n = 1,131 (target); 7,700 (non-target; data represent the average from two independent datasets; each box shows the first quartile, median, and third quartile; whiskers represent 1.5 × interquartile ranges; grey dots represent outliers; one-way ANOVA with Tukey's post hoc test, **P ≤ 2.2 × 10⁻¹⁶). h, m⁶A_m mRNAs show resistance to miR-155-mediated mRNA degradation. We tested if the identity of the first nucleotide affects the response of miR-155 target mRNAs to miR-155-mediated mRNA degradation. We observed that miR-155 target mRNAs that start with m⁶A_m show no significant suppression upon miR-155 transfection compared to non-target mRNAs that start with m⁶A_m. However, expression of miR-155 target mRNAs that start with A_m, C_m, G_m or U_m was significantly suppressed compared to non-target mRNAs that start with A_m, C_m, G_m or U_m. These data suggest that the presence m⁶A_m can reduce the silencing efficiency of a single microRNA in vivo (n = 1,714 versus 232 (m⁶A_m, non-target versus target); 953 versus 158 (A_m, non-target versus target); 1,848 versus 281 (C_m, non-target versus target); 1,394 versus 182 (G_m); 1,809 versus 278 (U_m, non-target versus target); 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 ≤ 0.05 non-target versus miR-155 target, **P ≤ 0.01 non-target versus miR-155 target, ***P ≤ 0.001 non-target versus miR-155 target).

Extended Data Figure 9. m⁶A_m-containing mRNAs are enriched for oxidative phosphorylation, metabolic pathways, and components of the RNA processing machinery

Gene Ontology (GO) analysis of m⁶A_m mRNAs. We used PANTHER overrepresentation test and Bonferroni correction with a P value threshold of <0.01. All annotated non-m⁶A_m-containing mRNAs (N_m) were used as the background gene list. m⁶A_m mRNAs are overrepresented in cellular pathways associated with oxidative phosphorylation and metabolism as well as mRNA processing and translation, suggesting that m⁶A_m controls cellular pathways by stabilizing specific populations of mRNAs.

Figure 1. FTO prefers m⁶A_m to m⁶A as a substrate

a , Modifications of the extended mRNA cap. The first nucleotide (here shown as adenosine) adjacent to the m⁷G and the 5′-to-5′ triphosphate (ppp) linker is subjected to 2′-O-methylation (orange) on the ribose, forming cap1. Cap1 can be further 2′-O-methylated at the second nucleotide to form cap2 (not depicted). 2′-O-methyladenosine (A_m) can be further converted to cap1m by N⁶-methylation (blue), which results in N⁶,2′-O-dimethyladenosine (m⁶A_m). b, FTO efficiently converts m⁶A_m to A_m. A synthetic oligonucleotide with a 5′-m⁷Gpppm⁶A_m (2 μM) was incubated with FTO (100 nM FTO, 1 h), which readily converted m⁶A_m to A_m (representative high-performance liquid chromatography (HPLC) track of n = 3 biological replicates). mAU, milli absorbance units. c, FTO preferentially demethylates m⁶A_m compared to m⁶A. An oligonucleotide with a 5′-m⁷Gpppm⁶A_m cap was mixed in an equimolar ratio with an oligonucleotide containing internal m⁶A. FTO (100 nM, 1 h) almost completely converted m⁶A_m to A_m. Demethylation of m⁶A was not detectable (representative HPLC track of n = 3 biological replicates). d, Michaelis-Menten kinetics of FTO for m⁶A_m and m⁶A. Owing to the increased activity of FTO with m⁶A_m compared to m⁶A, enzyme concentration was tenfold lower for m⁶A_m (20 nM FTO for m⁶A_m, 200 nM FTO for m⁶A). The data was normalized to enzyme concentration (m⁷Gpppm⁶A_m (blue), m⁷GpppACm⁶A (orange), internal m⁶A (green); n = 3 biological replicates; mean ± s.e.m; V₀ = initial reaction velocity).

Figure 2. m⁶A_m is the preferred substrate of FTO in vivo

a, FTO readily demethylates m⁶A_m in mRNA. Relative abundance of modified adenosines in mRNA caps derived from mRNA treated with FTO (1 μM, 1 h; representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, **P≤; 0.001). b, FTO expression decreases m⁶A_m in HEK293T cells. Relative abundance of modified adenosines in mRNA caps of HEK293T cells expressing GFP (Flag-GFP) or wild-type FTO (Flag-FTO) (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P ≤ 0.05). c, FTO knockdown increases m⁶A_m in HEK293T cells. Relative abundance of modified adenosines in mRNA caps of HEK293T cells transfected with scrambled siRNA (siCtrl) or siRNA directed against FTO (siFTO) (representative images shown; n = 3 biological replicates; mean ± s.e.m.; unpaired Student's t-test, *P≤ 0.05).

Figure 3. The presence of m⁶A_m is associated with increased mRNA half-life

a , mRNA stability is determined by the first encoded nucleotide in HEK293T cells. Cumulative distribution plot of the half-life for mRNAs that start with m⁶A_m, A_m, C_m, G_m and U_m (n = 2,515 (m⁶A_m); 762 (A_m); 1,442 (C_m); 1,119 (G_m); 1,486 (U_m); data represent the average from two independent datasets; 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 ≤ 2 × 10⁻⁸ versus m⁶A_m; N_m = A_m, C_m, G_m or U_m). b, mRNA expression level is influenced by the modification state of the first encoded nucleotide in HEK293T cells. Cumulative distribution plot of the expression for mRNAs that start with m⁶A_m, A_m, C_m, G_m and U_m (n = 2,536 (m⁶A_m); 1,063 (A_m); 2,098 (C_m); 1,577 (G_m); 2,071 (U_m); data represent the average from two independent datasets; 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 ≤ 2.2 × 10⁻¹⁶ versus m⁶A_m). c, FTO expression leads to a global decrease of m⁶A_m mRNA half-life in HEK293T cells. Changes in half-life of mRNAs containing either m⁶A_m or A_m in cells transfected with either Flag vector (Ctrl) or FTO with an N-terminal nuclear export signal (NES-FTO) (n = 2,049 (m⁶A_m); 951 (A_m); 1,442 (C_m); 1,119 (G_m); 1,486 (U_m); data represent the average from two independent datasets; 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 ≤ 4.6 × 10⁻³ versus m⁶A_m). d, FTO knockdown leads to a global increase of m⁶A_m mRNAs in HEK293T cells. Expression of mRNAs containing either m⁶A_m or A_m upon FTO knockdown (n = 3,410 (m⁶A_m); 1,355 (A_m); 2,636 (C_m); 1,994 (G_m); 2,558 (U_m); data represent the average from two independent mRNA expression datasets; 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 ≤ 7.4 × ⁻³ m⁶A_m versus A_m and U_m).

Figure 4. m⁶A_m mRNAs are resistant to DCP2-mediated decapping

a, Schematic representation of the DCP2 in vitro decapping assay. The 5′ end of oligonucleotides containing the indicated form of adenosine (A, A_m, m⁶A or m⁶A_m) was enzymatically capped with [α-³²P]-m⁷GTP. DCP2 causes the release of [α-³²P]-m⁷GDP, which is detected by TLC. b, N⁶-methylation of the cap-adjacent adenosine inhibits mRNA decapping in vitro. The presence of a 2′-O-methyl did not affect DCP2 activity relative to adenosine. However, addition of an N⁶-methyl group decreased decapping efficiency (n = 3 biological replicates; mean ± s.e.m.; two-way ANOVA with Tukey's post hoc test, **P≤ 0.01). c, DCP2 deficiency primarily increases expression of non-m⁶A_m mRNAs in the HEK293T cell transcriptome. Cumulative distribution plot of the expression of mRNAs that start with m⁶A_m, A_m, C_m, G_m and U_m (n = 3,287 (m⁶A_m); 2,350 (A_m); 3,963 (C_m); 3,540 (G_m); 3,496 (U_m); data represent the average from two independent datasets; 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 ≤ 2.2 × 10⁻¹⁶ versus m⁶A_m). d, m⁶A_m reduces mRNA susceptibility to microRNA-mediated degradation. Cumulative distribution plot of the expression of mRNAs that start with m⁶A_m, A_m, C_m, G_m or U_m (n = 2,090 (m⁶A_m); 623 (A_m); 1,109 (C_m); 852 (G_m); 1,322 (U_m); data represent the average from two independent datasets; 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≤2.2 × 10⁻¹⁶ versus m⁶A_m).