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NADP modulates RNA m6A methylation and adipogenesis via enhancing FTO activity

Nature Chemical Biology volume 16, pages 1394–1402 (2020)Cite this article

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An Author Correction to this article was published on 01 September 2023

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Abstract

Metabolism is often regulated by the transcription and translation of RNA. In turn, it is likely that some metabolites regulate enzymes controlling reversible RNA modification, such as N6-methyladenosine (m6A), to modulate RNA. This hypothesis is at least partially supported by the findings that multiple metabolic diseases are highly associated with fat mass and obesity-associated protein (FTO), an m6A demethylase. However, knowledge about whether and which metabolites directly regulate m6A remains elusive. Here, we show that NADP directly binds FTO, independently increases FTO activity, and promotes RNA m6A demethylation and adipogenesis. We screened a set of metabolites using a fluorescence quenching assay and NADP was identified to remarkably bind FTO. In vitro demethylation assays indicated that NADP enhances FTO activity. Furthermore, NADP regulated mRNA m6A via FTO in vivo, and deletion of FTO blocked NADP-enhanced adipogenesis in 3T3-L1 preadipocytes. These results build a direct link between metabolism and RNA m6A demethylation.

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Fig. 1: MARTFQ screening reveals that NADP directly binds FTO.
Fig. 2: NADP increases FTO activity in vitro.
Fig. 3: Intracellular NADP modulates RNA m6A methylation through FTO.
Fig. 4: The NADP/FTO axis increases adipogenesis in 3T3-L1 cells.
Fig. 5: NADP modulates RNA m6A methylation in vivo through FTO.

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Data availability

All sequencing data have been deposited to NCBI under accession no. PRJNA546061. Source Data are provided with this paper.

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Acknowledgements

We thank C. He at the University of Chicago for providing information for the expression and purification of FTO and ALKBH5. This study was supported by grants from the National Natural Science Foundation of China (NSFC nos. 81872310 and 81622040 to Q.Y.). This study was also supported by LiaoNing Revitalization Talents Program (XLYC1802058 to Q.Y.).

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Author notes

  1. These authors contributed equally: Lina Wang, Chengli Song, Na Wang.

Authors and Affiliations

  1. Institute of Cancer Stem Cell, DaLian Medical University, Dalian, Liaoning, China

    Lina Wang, Chengli Song, Na Wang, Songyu Li, Qiaoling Liu, Zhen Sun, Kai Wang & Qingkai Yang

  2. Department of Stem Cell and Regenerative Medicine, Southwest Hospital, Third Military Medical University (Army Medical University), Chongqing, China

    Shi-Cang Yu

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Contributions

Q.Y. and S.-C.Y. designed the experiments and wrote the paper. L.W., C.S. and N.W. performed most experiments. C.S. performed bioinformatics analyses. N.W. and S.L. raised the mice and performed the Oil red O staining and triglyceride assays. Q.L. expressed and purified the FTO and ALKBH5 proteins. N.W. and Z.S. maintained cells and performed immunoblotting analysis. K.W. constructed the plasmids.

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Correspondence to Shi-Cang Yu or Qingkai Yang.

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Extended data

Extended Data Fig. 1 MARTFQ screening reveals that NADP directly binds FTO.

a, Schematic showing MARTFQ assay. b, 3D structure of FTO protein containing tryptophan residues. The data for the FTO 3D structure were derived from NCBI accession code: 3LFM (Protein Data Bank). Red dots represent tryptophan residues in FTO protein. c, Coomassie staining of in vitro purified FTO protein. d, 3D structure of FTO. The data of FTO 3D structure were derived from accession code: 3LFM (Protein Data Bank). N terminal: blue; C terminal: red; 2KG: green. e, Coomassie staining of in vitro purified 6XHis-FTO-NT and 6XHis-FTO-CT proteins. f, Quenching of the fluorescence of FTO-NT (left) and FTO-CT (right) proteins by NADPH at different molar ratios. g, The modified Stern–Volmer curves for estimating the K values of FTO-NT (18.5 x 103 M-1) and FTO-CT (30.2 x 103 M-1). h, Immunoblotting analyses of the cell lysates from HEK293 cells transfected with pCDNA3 Flag-FTO (WT), pCI-Neo Flag-FTO-NT or pCI-Neo Flag-FTO-CT plasmid. i,j, DARTS (i) and CETSA (j) analyses of the abilities of NADPH to bind FTO-NT and FTO-CT in HEK293 cells transfected with pCI-Neo Flag-FTO-NT or pCI-Neo Flag-FTO-CT plasmid. Immunoblotting was performed using anti-Flag antibody.

Source data

Extended Data Fig. 2 NADP increases the FTO activity in vitro.

a, Schematic showing the in vitro m6A demethylation assay. b, Base ion mass transitions of A and m6A in standard compounds and mRNAs at different collision energies. c, Kinetic curves of FTO in the presence of 2 mM Vc. d, MS analyses of m6A in the post-reaction mixtures as described in Fig. 2a. e,f, Dot blotting analyses of m6A in the FTO post-reaction mixtures as described in Fig. 2a (e) and Fig. 2b (f).

Extended Data Fig. 3 NADP was not stoichiometrically consumed in the reaction catalyzed by FTO.

a, MS analyses of the compounds as noted in the post-reaction mixtures containing compound as noted. NADPH, NADP, NADH, NAD or Vc at 10 µM was separately added into the mixtures (10 µM ssRNA, 0.5 µM FTO, 300 µM Fe2+ and 300 µM 2KG). Boiled FTO was used as the negative control. Resultant mixtures were incubated at 37 °C for 90 min without heatin. b, Demethylation of m6A in the FTO reaction mixtures containing varied concentration of Vc or DHA for 10 min. c, Demethylation of m6A in the FTO reaction mixtures incubated for varied times in the presence of 0.5 mM of Vc or DHA. d, Coomassie staining of in vitro purified ALKBH5 protein. e, Kinetic curves of m6A ssRNA for ALKBH5 in the presence of 2 mM Vc. f, MS analyses of m6A in the ALKBH5 post-reaction mixtures described in Fig. 2c. g, Demethylation of m6A in the ALKBH5 reaction mixtures containing high concentrations of the cofactor as noted.

Extended Data Fig. 4 Intracellular NADP modulates RNA m6A methylation through FTO.

a,b, Total concentration (a) and reduced/oxidized ratio (b) of NADP in the cells. c,d, Total concentration (c) and reduced/oxidized ratio (d) of NAD in the cells. e, The NADP and NAD levels in the cells treated with 300 µM NADPH, NADP+, NADH or NAD+ for 4 hrs. f,g, Total concentration and reduced/oxidized ratio of NADP and NAD in the fourth fat pads (f) and livers (g) of mice treated with HFD for 6 hrs or IP injection of glucose for 4 hrs. h, The NADP and NAD levels in liver of mice described in (g). i, The Vc levels in the cells described in Fig. 3c. j,k, Dot blot analyses of DNA 5hmdC (j) and RNA m6A (k) levels in the cells treated with 300 µM Vc or NADPH for 4 hrs.

Source data

Extended Data Fig. 5 NADP modulates RNA m6A methylation via FTO.

a, Schematic showing the synthesis of NADP and NAD. b, Protein levels in HEK293 cells treated as described in Fig. 3e. c, The NADP and NAD levels in the cells treated as described in Fig. 3e. d, Protein levels in HEK293 cells treated as described in Fig. 3g. e, The NADP and NAD levels in the cells treated as described in Fig. 3g. f, The NADP and NAD levels in the cells treated as described in Fig. 3i. g, The m6A levels in the cells treated with NADPH and/or 6AN for 6 hrs. h, Protein levels in the cells treated with siRNA as noted. i, The m6A levels in HEK293 cells treated as described in Fig. 3j.

Source data

Extended Data Fig. 6 Identification of the NADP\FTO-regulated m6A methylated genes by anti-m6A MeRIP-seq.

a, Schematic showing anti-m6A MeRIP-seq of control (Ctrl) and knockdown (KD) 3T3-L1 cells treated with 6AN for 8 hrs or NADPH for 4 hrs. b,c, Volcano plots of p value versus FC of 6AN/Mock (b) and NADPH/Mock (c) in Ctrl and KD cells. Red plot: substantially upregulated peak (SUP) with p < 0.05 and FC > 1.5; green plot: substantially downregulated peak (SDP) with p < 0.05 and FC < 0.66. d,e, Volcano plots of p value versus FC in CDS, 3’UTR and 5’UTR regions of 6AN/Mock (d) and NADPH/Mock (e) in Ctrl and KD cells. f, Ratio of SUP number versus SDP number in Ctrl and KD cells treated with 6AN or NADPH. g, Ratio of SUP/SDP in CDS and 5’UTR. h, The distribution curves of treatment/Mock peaks in Ctrl and KD cells.

Extended Data Fig. 7 Enrichment and validation analyses of NFRM genes.

a, Venn diagram of 265 NFRM genes showing the overlap of the significant peaks in Ctrl and KD cells treated with 6AN or NADPH. b, Protein-protein interaction network analysis of NFRM genes. The molecular complex detection (MCODE) algorithm was applied to identify densely connected network components using Metascape (metascape.org). Identified MCODE networks are gathered and shown by Cytoscape (www.cytoscape.org). c, Top 5 protein complexes with significant p value of enrichment. d,e, Reactome (d) and GO (e) enrichment analyses of NFRM genes. f, The normalized ΔCt levels of 17 selected genes in 3T3-L1 cells treated with Mock, FB23-2 (FTO inhibitor), NADPH or NADPH+FB23-2 for 4 hrs. The ΔCt values were normalized to Gapdh. g,h, The qRT-PCR analyses of anti-Fto PAR-CLIP (g) and anti-m6A MeRIP (h) RNAs. The RNA level in IgG IP was taken as 1. i,j, The relative RNA levels in 3T3-L1 cells treated with actinomycin D for 3 (i) or 6 (j) hrs. 3T3-L1 cells were treated with Mock, FB23-2, NADPH or NADPH+FB23-2 for 4 hrs. Then actinomycin D was applied to stop transcription. The RNA level in the Mock cells treated with actinomycin D for 0 h was taken as 1.

Source data

Extended Data Fig. 8 NADP\FTO axis increases adipogenesis in 3T3-L1 cells.

a,b, Oil Red O staining of 3T3-L1 cells on day 6 (a) and day 8 (D8) (b). 3T3-L1 cells were treated with siRNAi against control, Fto, Alkbh5 or Mettl3 on day -2 (D-2). After 2 days, 3T3-L1 preadipocytes were induced to differentiate by MDI cocktail on D0. Resultant cells were stained by Oil Red O. Scale bar = 100 μm. c, The NADP and NAD levels in control and Fto knockdown 3T3-L1 cells treated with NADPH. d, Oil Red O staining and triglyceride analyses of 3T3-L1 cells pretreated with or without siFto were treated with 300 μM of NADPH or NADP+ prior to induction. e, Oil Red O staining and triglyceride analyses of 3T3-L1 cells treated with NADPH, NADP+, and/or FB23-2 (20 μM) prior to induction. f, Schematic showing the treatment of FTO inhibitor for the time as noted. g, Oil Red O staining and triglyceride analyses of 3T3-L1 cells treated as described in (f). h, Oil Red O staining and triglyceride analyses of 3T3-L1 cells treated with 6AN, NADPH and/or FB23-2 for 6 hrs prior to induction.

Source data

Extended Data Fig. 9 The impact of NADP for adipogenesis is dependent on the catalytic activity of FTO.

a, The NADP and NAD levels in control and Fto knockdown 3T3-L1 cells treated with 0.5 μM insulin and/or 500 μM 6AN for 6 hrs. b, The m6A levels in control and Fto knockdown 3T3-L1 cells treated with insulin and/or 6AN for 6 hrs. c, The m6A levels in 3T3-L1 cells treated with FB23-2, insulin and/or 6AN as noted for 6 hrs. d, Oil Red O staining and triglyceride analyses of 3T3-L1 cells treated with FB23-2, insulin and/or 6AN prior to induction. e, Immunoblotting analyses of the cell lysates from mouse Fto knockdown 3T3-L1 cells infected with adenovirus encoding human wild type (Ad FTO-WT) or catalytic inactive mutant (Ad FTO-Mut) FTO. f, The m6A levels in control and Fto knockdown 3T3-L1 cells treated with adenoviral particles and NADPH. The 3T3-L1 cells were treated with siRNAs and adenoviruses as noted. The resultant cells were treated with NADPH for 6 hrs. g,h, Oil Red O staining and triglyceride analyses of 3T3-L1 cells treated with siRNAs, adenoviral particles, NADPH (g) or insulin (h) as noted.

Source data

Extended Data Fig. 10 NADP modulates RNA m6A methylation in vivo through FTO.

a, PCR analyses of the genomic DNA samples from WT and KO mice using the primers as described in Supplementary Table 1. b, Representative images of WT and KO mice at different ages. c, Body weights of male WT and KO mice at different ages. WT mice, n = 14; KO mice, n = 11. d, Haematoxylin and eosin staining of white adipose tissue (WAT) of WT and KO mice treated with HFD for 12 weeks. e, The total concentrations of Vc, NAD and NADP in the livers and fourth fat pads of mice. n = 6 mice. f-h, The Vc (f), NAD(P) (g) and 5mdC (h) levels in the fat pads described in Fig. 5g. i-m, The Vc (i), NAD(P) (j), 5hmdC (k), 5mdC (l) and m6A (m) levels in the liver as described in Fig. 5g.

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Wang, L., Song, C., Wang, N. et al. NADP modulates RNA m6A methylation and adipogenesis via enhancing FTO activity. Nat Chem Biol 16, 1394–1402 (2020). https://doi.org/10.1038/s41589-020-0601-2

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