N6-methyladenosine (m6A) is the most abundant base modification found in messenger RNAs (mRNAs). The discovery of FTO as the first m6A mRNA demethylase established the concept of reversible RNA modification. Here, we present a comprehensive transcriptome-wide analysis of RNA demethylation and uncover FTO as a potent regulator of nuclear mRNA processing events such as alternative splicing and 3΄ end mRNA processing. We show that FTO binds preferentially to pre-mRNAs in intronic regions, in the proximity of alternatively spliced (AS) exons and poly(A) sites. FTO knockout (KO) results in substantial changes in pre-mRNA splicing with prevalence of exon skipping events. The alternative splicing effects of FTO KO anti-correlate with METTL3 knockdown suggesting the involvement of m6A. Besides, deletion of intronic region that contains m6A-linked DRACH motifs partially rescues the FTO KO phenotype in a reporter system. All together, we demonstrate that the splicing effects of FTO are dependent on the catalytic activity in vivo and are mediated by m6A. Our results reveal for the first time the dynamic connection between FTO RNA binding and demethylation activity that influences several mRNA processing events.

CEITEC Central European Institute of Technology Masaryk University Brno 62500 Czech Republic

MRC Human Genetics Unit MRC IGMM University of Edinburgh Western General Hospital Crewe Road Edinburgh EH4 2XU UK

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Meyer K.D., Saletore Y., Zumbo P., Elemento O., Mason C.E., Jaffrey S.R.. Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell. 2012; 149:1635–1646. PubMed PMC

Dominissini D., Moshitch-Moshkovitz S., Schwartz S., Salmon-Divon M., Ungar L., Osenberg S., Cesarkas K., Jacob-Hirsch J., Amariglio N., Kupiec M. et al. . Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq. Nature. 2012; 485:201–206. PubMed

Wang Y., Li Y., Toth J.I., Petroski M.D., Zhang Z., Zhao J.C.. N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 2014; 16:191–198. PubMed PMC

Wang X., Lu Z., Gomez A., Hon G.C., Yue Y., Han D., Fu Y., Parisien M., Dai Q., Jia G. et al. . N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014; 505:117–120. PubMed PMC

Schwartz S., Mumbach M.R., Jovanovic M., Wang T., Maciag K., Bushkin G.G., Mertins P., Ter-Ovanesyan D., Habib N., Cacchiarelli D. et al. . Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep. 2014; 8:284–296. PubMed PMC

Ping X.L., Sun B.F., Wang L., Xiao W., Yang X., Wang W.J., Adhikari S., Shi Y., Lv Y., Chen Y.S. et al. . Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014; 24:177–189. PubMed PMC

Liu J., Yue Y., Han D., Wang X., Fu Y., Zhang L., Jia G., Yu M., Lu Z., Deng X. et al. . A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat. Chem. Biol. 2014; 10:93–95. PubMed PMC

Batista P.J., Molinie B., Wang J., Qu K., Zhang J., Li L., Bouley D.M., Lujan E., Haddad B., Daneshvar K. et al. . m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell. 2014; 15:707–719. PubMed PMC

Adams J.M., Cory S.. Modified nucleosides and bizarre 5′-termini in mouse myeloma mRNA. Nature. 1975; 255:28–33. PubMed

Dubin D.T., Taylor R.H.. The methylation state of poly A-containing messenger RNA from cultured hamster cells. Nucleic Acids Res. 1975; 2:1653–1668. PubMed PMC

Furuichi Y., Morgan M., Shatkin A.J., Jelinek W., Salditt-Georgieff M., Darnell J.E.. Methylated, blocked 5 termini in HeLa cell mRNA. Proc. Natl. Acad. Sci. U.S.A. 1975; 72:1904–1908. PubMed PMC

Moyer S.A., Abraham G., Adler R., Banerjee A.K.. Methylated and blocked 5′ termini in vesicular stomatitis virus in vivo mRNAs. Cell. 1975; 5:59–67. PubMed

Wei C., Gershowitz A., Moss B.. N6, O2′-dimethyladenosine a novel methylated ribonucleoside next to the 5′ terminal of animal cell and virus mRNAs. Nature. 1975; 257:251–253. PubMed

Wei C.M., Gershowitz A., Moss B.. Methylated nucleotides block 5′ terminus of HeLa cell messenger RNA. Cell. 1975; 4:379–386. PubMed

Schibler U., Kelley D.E., Perry R.P.. Comparison of methylated sequences in messenger RNA and heterogeneous nuclear RNA from mouse L cells. J. Mol. Biol. 1977; 115:695–714. PubMed

Munns T.W., Liszewski M.K., Sims H.F.. Characterization of antibodies specific for N6-methyladenosine and for 7-methylguanosine. Biochemistry. 1977; 16:2163–2168. PubMed

Linder B., Grozhik A.V., Olarerin-George A.O., Meydan C., Mason C.E., Jaffrey S.R.. Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods. 2015; 12:767–772. PubMed PMC

Xiao W., Adhikari S., Dahal U., Chen Y.S., Hao Y.J., Sun B.F., Sun H.Y., Li A., Ping X.L., Lai W.Y. et al. . Nuclear m(6)A reader YTHDC1 regulates mRNA splicing. Mol. Cell. 2016; 61:507–519. PubMed

Merkestein M., Laber S., McMurray F., Andrew D., Sachse G., Sanderson J., Li M., Usher S., Sellayah D., Ashcroft F.M. et al. . FTO influences adipogenesis by regulating mitotic clonal expansion. Nat. Commun. 2015; 6:6792. PubMed PMC

Liu N., Dai Q., Zheng G., He C., Parisien M., Pan T.. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015; 518:560–564. PubMed PMC

Zhao X., Yang Y., Sun B.F., Shi Y., Yang X., Xiao W., Hao Y.J., Ping X.L., Chen Y.S., Wang W.J. et al. . FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 2014; 24:1403–1419. PubMed PMC

Fustin J.M., Doi M., Yamaguchi Y., Hida H., Nishimura S., Yoshida M., Isagawa T., Morioka M.S., Kakeya H., Manabe I. et al. . RNA-methylation-dependent RNA processing controls the speed of the circadian clock. Cell. 2013; 155:793–806. PubMed

Meyer K.D., Patil D.P., Zhou J., Zinoviev A., Skabkin M.A., Elemento O., Pestova T.V., Qian S.B., Jaffrey S.R.. 5′ UTR m(6)A promotes Cap-independent translation. Cell. 2015; 163:999–1010. PubMed PMC

Wang X., Zhao B.S., Roundtree I.A., Lu Z., Han D., Ma H., Weng X., Chen K., Shi H., He C.. N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015; 161:1388–1399. PubMed PMC

Zhou J., Wan J., Gao X., Zhang X., Jaffrey S.R., Qian S.B.. Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature. 2015; 526:591–594. PubMed PMC

Lin S., Choe J., Du P., Triboulet R., Gregory R.I.. The m(6)A methyltransferase METTL3 promotes translation in human cancer cells. Mol. Cell. 2016; 62:335–345. PubMed PMC

Zheng G.Q., Dahl J.A., Niu Y.M., Fedorcsak P., Huang C.M., Li C.J., Vagbo C.B., Shi Y., Wang W.L., Song S.H. et al. . ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell. 2013; 49:18–29. PubMed PMC

Wang P., Doxtader K.A., Nam Y.. Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases. Mol. Cell. 2016; 63:306–317. PubMed PMC

Wang X., Feng J., Xue Y., Guan Z., Zhang D., Liu Z., Gong Z., Wang Q., Huang J., Tang C. et al. . Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature. 2016; 534:575–578. PubMed

Harper J.E., Miceli S.M., Roberts R.J., Manley J.L.. Sequence specificity of the human mRNA N6-adenosine methylase in vitro. Nucleic Acids Res. 1990; 18:5735–5741. PubMed PMC

Aguilo F., Zhang F., Sancho A., Fidalgo M., Di Cecilia S., Vashisht A., Lee D.F., Chen C.H., Rengasamy M., Andino B. et al. . Coordination of m(6)A mRNA methylation and gene transcription by ZFP217 regulates pluripotency and reprogramming. Cell Stem Cell. 2015; 17:689–704. PubMed PMC

Ke S., Alemu E.A., Mertens C., Gantman E.C., Fak J.J., Mele A., Haripal B., Zucker-Scharff I., Moore M.J., Park C.Y. et al. . A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation. Genes Dev. 2015; 29:2037–2053. PubMed PMC

Jia G.F., Fu Y., Zhao X., Dai Q., Zheng G.Q., Yang Y., Yi C.Q., Lindahl T., Pan T., Yang Y.G. et al. . N6-Methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO (vol 7, pg 885, 2011). Nat. Chem. Biol. 2012; 8:1008–1008. PubMed PMC

Zheng G., Dahl J.A., Niu Y., Fedorcsak P., Huang C.M., Li C.J., Vagbo C.B., Shi Y., Wang W.L., Song S.H. et al. . ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell. 2013; 49:18–29. PubMed PMC

Gulati P., Avezov E., Ma M., Antrobus R., Lehner P., O’Rahilly S., Yeo G.S.. Fat mass and obesity-related (FTO) shuttles between the nucleus and cytoplasm. Biosci. Rep. 2014; 34:e00144. PubMed PMC

Gulati P., Cheung M.K., Antrobus R., Church C.D., Harding H.P., Tung Y.C., Rimmington D., Ma M., Ron D., Lehner P.J. et al. . Role for the obesity-related FTO gene in the cellular sensing of amino acids. Proc. Natl. Acad. Sci. U.S.A. 2013; 110:2557–2562. PubMed PMC

Zhang M., Zhang Y., Ma J., Guo F., Cao Q., Zhang Y., Zhou B., Chai J., Zhao W., Zhao R.. The demethylase activity of FTO (fat mass and obesity associated protein) is required for preadipocyte differentiation. PLoS One. 2015; 10:e0133788. PubMed PMC

Zou S., Toh J.D., Wong K.H., Gao Y.G., Hong W., Woon E.C.. N(6)-Methyladenosine: a conformational marker that regulates the substrate specificity of human demethylases FTO and ALKBH5. Sci. Rep. 2016; 6:25677. PubMed PMC

Frayling T.M., Timpson N.J., Weedon M.N., Zeggini E., Freathy R.M., Lindgren C.M., Perry J.R., Elliott K.S., Lango H., Rayner N.W. et al. . A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity. Science. 2007; 316:889–894. PubMed PMC

Smemo S., Tena J.J., Kim K.H., Gamazon E.R., Sakabe N.J., Gomez-Marin C., Aneas I., Credidio F.L., Sobreira D.R., Wasserman N.F. et al. . Obesity-associated variants within FTO form long-range functional connections with IRX3. Nature. 2014; 507:371–375. PubMed PMC

Claussnitzer M., Dankel S.N., Kim K.H., Quon G., Meuleman W., Haugen C., Glunk V., Sousa I.S., Beaudry J.L., Puviindran V. et al. . FTO obesity variant circuitry and adipocyte browning in humans. N. Engl. J. Med. 2015; 373:895–907. PubMed PMC

Shen B., Zhang W.S., Zhang J., Zhou J.K., Wang J.Y., Chen L., Wang L., Hodgkins A., Iyer V., Huang X.X. et al. . Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods. 2014; 11:399. PubMed

Cong L., Ran F.A., Cox D., Lin S.L., Barretto R., Habib N., Hsu P.D., Wu X.B., Jiang W.Y., Marraffini L.A. et al. . Multiplex genome engineering using CRISPR/Cas systems. Science. 2013; 339:819–823. PubMed PMC

Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F.. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 2013; 8:2281–2308. PubMed PMC

Konig J., Zarnack K., Rot G., Curk T., Kayikci M., Zupan B., Turner D.J., Luscombe N.M., Ule J.. iCLIP–transcriptome-wide mapping of protein-RNA interactions with individual nucleotide resolution. J. Vis. Exp. 2011; 50:2638. PubMed PMC

Ule J., Jensen K., Mele A., Darnell R.B.. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods. 2005; 37:376–386. PubMed

Kim D., Pertea G., Trapnell C., Pimentel H., Kelley R., Salzberg S.L.. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 2013; 14:R36. PubMed PMC

Love M.I., Huber W., Anders S.. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15:550. PubMed PMC

Anders S., Reyes A., Huber W.. Detecting differential usage of exons from RNA-seq data. Genome Res. 2012; 22:2008–2017. PubMed PMC

Katz Y., Wang E.T., Airoldi E.M., Burge C.B.. Analysis and design of RNA sequencing experiments for identifying isoform regulation. Nat. Methods. 2010; 7:1009–1015. PubMed PMC

Sims D., Ilott N.E., Sansom S.N., Sudbery I.M., Johnson J.S., Fawcett K.A., Berlanga-Taylor A.J., Luna-Valero S., Ponting C.P., Heger A.. CGAT: computational genomics analysis toolkit. Bioinformatics. 2014; 30:1290–1291. PubMed PMC

Young M.D., Wakefield M.J., Smyth G.K., Oshlack A.. Gene ontology analysis for RNA-seq: accounting for selection bias. Genome Biol. 2010; 11:R14. PubMed PMC

Quinlan A.R., Hall I.M.. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26:841–842. PubMed PMC

Durinck S., Bullard J., Spellman P.T., Dudoit S.. GenomeGraphs: integrated genomic data visualization with R. BMC Bioinformatics. 2009; 10:2. PubMed PMC

Xia Z., Donehower L.A., Cooper T.A., Neilson J.R., Wheeler D.A., Wagner E.J., Li W.. Dynamic analyses of alternative polyadenylation from RNA-seq reveal a 3′-UTR landscape across seven tumour types. Nat. Commun. 2014; 5:5274. PubMed PMC

Kishore S., Khanna A., Stamm S.. Rapid generation of splicing reporters with pSpliceExpress. Gene. 2008; 427:104–110. PubMed PMC

Schneider C.A., Rasband W.S., Eliceiri K.W.. NIH Image to ImageJ: 25 years of image analysis. Nat. Methods. 2012; 9:671–675. PubMed PMC

Gruber A.J., Schmidt R., Gruber A.R., Martin G., Ghosh S., Belmadani M., Keller W., Zavolan M.. A comprehensive analysis of 3′ end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation. Genome Res. 2016; 26:1145–1159. PubMed PMC

Martin G., Gruber A.R., Keller W., Zavolan M.. Genome-wide analysis of pre-mRNA 3′ end processing reveals a decisive role of human cleavage factor I in the regulation of 3′ UTR length. Cell Rep. 2012; 1:753–763. PubMed

Mauer J., Luo X., Blanjoie A., Jiao X., Grozhik A.V., Patil D.P., Linder B., Pickering B.F., Vasseur J.J., Chen Q. et al. . Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature. 2016; 541:371–375. PubMed PMC

Castello A., Fischer B., Eichelbaum K., Horos R., Beckmann B.M., Strein C., Davey N.E., Humphreys D.T., Preiss T., Steinmetz L.M. et al. . Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell. 2012; 149:1393–1406. PubMed

Baltz A.G., Munschauer M., Schwanhausser B., Vasile A., Murakawa Y., Schueler M., Youngs N., Penfold-Brown D., Drew K., Milek M. et al. . The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Molecular Cell. 2012; 46:674–690. PubMed

Penn J.K., Graham P., Deshpande G., Calhoun G., Chaouki A.S., Salz H.K., Schedl P.. Functioning of the Drosophila Wilms'-tumor-1-associated protein homolog, Fl(2)d, in Sex-lethal-dependent alternative splicing. Genetics. 2008; 178:737–748. PubMed PMC

Haussmann I.U., Bodi Z., Sanchez-Moran E., Mongan N.P., Archer N., Fray R.G., Soller M.. m6A potentiates Sxl alternative pre-mRNA splicing for robust Drosophila sex determination. Nature. 2016; 540:301–304. PubMed

Kan L., Grozhik A.V., Vedanayagam J., Patil D.P., Pang N., Lim K.S., Huang Y.C., Joseph B., Lin C.J., Despic V. et al. . The m6A pathway facilitates sex determination in Drosophila. Nat. Commun. 2017; 8:15737. PubMed PMC

Lence T., Akhtar J., Bayer M., Schmid K., Spindler L., Ho C.H., Kreim N., Andrade-Navarro M.A., Poeck B., Helm M. et al. . m6A modulates neuronal functions and sex determination in Drosophila. Nature. 2016; 540:242–247. PubMed

Ke S., Pandya-Jones A., Saito Y., Fak J.J., Vagbo C.B., Geula S., Hanna J.H., Black D.L., Darnell J.E. Jr, Darnell R.B.. m6A mRNA modifications are deposited in nascent pre-mRNA and are not required for splicing but do specify cytoplasmic turnover. Genes Dev. 2017; 31:990–1006. PubMed PMC

Fu Y., Jia G., Pang X., Wang R.N., Wang X., Li C.J., Smemo S., Dai Q., Bailey K.A., Nobrega M.A. et al. . FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat. Commun. 2013; 4:1798. PubMed PMC

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