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The detection and functions of RNA modification m6A based on m6A writers and erasers

  • Wei Zhang
    Affiliations
    Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
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  • Yang Qian
    Affiliations
    Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
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  • Guifang Jia
    Correspondence
    For correspondence: Guifang Jia
    Affiliations
    Synthetic and Functional Biomolecules Center, Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, College of Chemistry and Molecular Engineering, Peking University, Beijing, China
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Open AccessPublished:July 16, 2021DOI:https://doi.org/10.1016/j.jbc.2021.100973
      N6-methyladenosine (m6A) is the most frequent chemical modification in eukaryotic mRNA and is known to participate in a variety of physiological processes, including cancer progression and viral infection. The reversible and dynamic m6A modification is installed by m6A methyltransferase (writer) enzymes and erased by m6A demethylase (eraser) enzymes. m6A modification recognized by m6A binding proteins (readers) regulates RNA processing and metabolism, leading to downstream biological effects such as promotion of stability and translation or increased degradation. The m6A writers and erasers determine the abundance of m6A modifications and play decisive roles in its distribution and function. In this review, we focused on m6A writers and erasers and present an overview on their known functions and enzymatic molecular mechanisms, showing how they recognize substrates and install or remove m6A modifications. We also summarize the current applications of m6A writers and erasers for m6A detection and highlight the merits and drawbacks of these available methods. Lastly, we describe the biological functions of m6A in cancers and viral infection based on research of m6A writers and erasers and introduce new assays for m6A functionality via programmable m6A editing tools.

      Keywords

      Abbreviations:

      3′ UTR (3′ untranslated region), 5 mC (5-methylcytosine), α-KG (α-ketoglutarate), Ac4C (N4-acetylcytidine), AML (acute myeloid leukemia), dm6A (N6-dithiolsitolmethyladenosine), DTT (dithiothreitol), FTO (fat obesity-associated protein), GSC (glioblastoma stem cell), HCV (Hepatitis C virus), IFN (interferon), m6A (N6-methyladenosine), miCLIP (m6A individual-nucleotide-resolution cross-linking and immunoprecipitation), miRNA (microRNA), mRNA (messenger RNA), OGDH (α-KG dehydrogenase), PAM (protospacer adjacent motif), RIG-I (retinoic-acid-inducible gene I), RIP (RNA immunoprecipitation), rRNA (ribosomal RNA), SAM (S-adenosyl methionine), SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), SFPQ (splicing factor proline and glutamine-rich), snRNA (small nuclear RNA), tRNA (transfer RNA), WTAP (Wilms’ tumor 1-associating protein), XIST (X-inactive specific transcript), ZIKV (Zika virus)
      RNA plays a central role in central dogma of molecular biology and is responsible for transmitting the genetic information encoded by DNA into functional proteins. There are different types of RNA molecules that are involved in the regulation of several biological processes, including messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), microRNA (miRNA), long noncoding RNA (lncRNA), etc. Analogous to DNA or histone modifications (
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      Figure thumbnail gr1
      Figure 1Scheme overview of m6A machinery in mammals. m6A is deposited and removed by m6A methyltransferase complex (writers) and demethylases (erasers) in the nucleus respectively to realize dynamic regulation. m6A-binding proteins (m6A readers) can recognize and bind m6A-modified mRNA to perform various biological functions such as increasing translation efficiency and degrading RNA.
      The development of m6A detection technology is of great importance for studying the biological functions of m6A. Mass spectrometry technology can identify changes in the overall content of m6A in cells, and m6A antibody-based high-throughput sequencing methods help us determine the distribution of m6A on transcripts (
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      ). However, the reliability and completeness of the results still need to be improved with more precise methods. In the past 2 years, the combination of m6A writers and erasers with CRISPR/Cas technology has achieved the programmable m6A editing, which provides benign verifying and regulating tools for deeply studying the functions of m6A (
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      In this review, we mainly focus on m6A writers and erasers. We will summarize the identification and enzymatic molecular mechanisms of m6A writers and erasers and compare m6A writers- and erasers-assisted m6A detection methods with traditional antibody-based methods. m6A writers and erasers regulate plenty of physiological and pathological processes, and here we will elaborate the recent findings about the regulatory roles of m6A writers and erasers in pathological processes, especially in viral infection and cancer progression. Finally, we will introduce the approaches for programmable m6A editing developed through the conjugation of m6A writers or erasers with CRISPR/Cas system, which could be widely used for m6A functional study.

      m6A writers and erasers

      m6A writers

      The m6A methyltransferase complex for installing the majority of mRNA m6A modifications

      m6A methyltransferase complex was first purified and characterized from the nuclear extracts of HeLa cells in 1990s (
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      ). The first catalytic subunit METTL3 was identified in 1997 (
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      • Rottman F.
      Purification and cDNA cloning of the AdoMet-binding subunit of the human mRNA (N6-adenosine)-methyltransferase.
      ). METTL3 contains an S-adenosyl methionine (SAM) binding domain and a DPPW motif (Asn-Pro-Pro-Trp) for transferring the methyl group from the SAM to the N6 position of the targeted adenosine (Fig. 2, A and B). Later, METTL14 was identified as the second core component of the mRNA m6A methyltransferase complex, sharing around 43% sequence identity with METTL3 based on sequence alignment analysis (
      • Yue Y.
      • Liu J.
      • He C.
      RNA N6-methyladenosine methylation in post-transcriptional gene expression regulation.
      ,
      • Liu J.Z.
      • Yue Y.N.
      • Han D.L.
      • Wang X.
      • Fu Y.
      • Zhang L.
      • Jia G.F.
      • Yu M.
      • Lu Z.K.
      • Deng X.
      • Dai Q.
      • Chen W.Z.
      • He C.
      A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
      ). Biochemical studies show that METTL14 and METTL3 form a heterodimer and exhibit higher m6A installation activity than METTL3 alone (
      • Liu J.Z.
      • Yue Y.N.
      • Han D.L.
      • Wang X.
      • Fu Y.
      • Zhang L.
      • Jia G.F.
      • Yu M.
      • Lu Z.K.
      • Deng X.
      • Dai Q.
      • Chen W.Z.
      • He C.
      A METTL3–METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation.
      ). The crystal structure analysis indicates that only METTL3 can bind SAM, and METTL14 structurally supports METTL3 by providing an RNA binding scaffold, which substantially enhances its methylation efficiency as a result (
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.Y.
      • Zhang D.L.
      • Liu Z.
      • Gong Z.
      • Wang Q.
      • Huang J.B.
      • Tang C.
      • Zou T.T.
      • Yin P.
      Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex.
      ,
      • Schöller E.
      • Weichmann F.
      • Treiber T.
      • Ringle S.
      • Treiber N.
      • Flatley A.
      • Feederle R.
      • Bruckmann A.
      • Meister G.
      Interactions, localization, and phosphorylation of the m6A generating METTL3–METTL14–WTAP complex.
      ,
      • Śledź P.
      • Jinek M.
      Structural insights into the molecular mechanism of the m6A writer complex.
      ) (Fig. 2A).
      Figure thumbnail gr2
      Figure 2The working mechanisms of m6A writers and erasers. A, crystal analysis indicates that METTL3 possesses an SAM-binding pocket and DPPL motif for m6A deposition and can form a stable heterodimer with METTL14. B, proposed mechanism of methyl transfer catalyzed by METTL3. C, proposed mechanisms of FTO toward different substrates. D, differential oxidation steps of FTO and ALKBH5 toward m6A. E, differential behavior of FTO and ALKBH5 in the nucleobase recognition region.
      METTL3 and METTL14 are key core subunits of m6A methyltransferase complex and play pivotal roles in different biological processes, including embryonic development and neurogenesis (
      • Batista P.J.
      • Molinie B.
      • Wang J.
      • Qu K.
      • Zhang J.
      • Li L.
      • Bouley D.M.
      • Lujan E.
      • Haddad B.
      • Daneshvar K.
      m6A RNA modification controls cell fate transition in mammalian embryonic stem cells.
      ,
      • Yoon K.-J.
      • Ringeling F.R.
      • Vissers C.
      • Jacob F.
      • Pokrass M.
      • Jimenez-Cyrus D.
      • Su Y.
      • Kim N.-S.
      • Zhu Y.
      • Zheng L.
      Temporal control of mammalian cortical neurogenesis by m6A methylation.
      ). METTL3−/− mice exhibit early embryonic lethality (
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • Hershkovitz V.
      • Peer E.
      • Mor N.
      • Manor Y.S.
      • Ben-Haim M.S.
      • Eyal E.
      • Yunger S.
      • Pinto Y.
      • Jaitin D.A.
      • et al.
      m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.
      ), and the depletion of DmIME4 (METTL3 homolog) leads to gametogenic defects in Drosophila (
      • Hongay C.F.
      • Orr-Weaver T.L.
      Drosophila Inducer of MEiosis 4 (IME4) is required for Notch signaling during oogenesis.
      ). Knockout of METTL14 and METTL3 in embryonic mouse brains can both prolong the cell cycle of radial glia cells and extend cortical neurogenesis into postnatal stages in an m6A-dependent manner (
      • Yoon K.-J.
      • Ringeling F.R.
      • Vissers C.
      • Jacob F.
      • Pokrass M.
      • Jimenez-Cyrus D.
      • Su Y.
      • Kim N.-S.
      • Zhu Y.
      • Zheng L.
      Temporal control of mammalian cortical neurogenesis by m6A methylation.
      ).
      Wilms’ tumor 1-associating protein (WTAP) has been identified as the third subunit of mRNA m6A methyltransferase complex and classified as a splicing factor that binds to the Wilms’ tumor 1 protein in mammals that plays a key role in embryonic development (
      • Little N.A.
      • Hastie N.D.
      • Davies R.C.
      Identification of WTAP, a novel Wilms’ tumour 1-associating protein.
      ,
      • Small T.W.
      • Pickering G.J.
      Nuclear degradation of Wilms tumor 1-associating protein and survivin splice variant switching underlie IGF-1-mediated survival.
      ). While WTAP has no catalytical activity toward RNA targets, it can assist the METTL3-METTL14 heterodimer to locate in nuclear speckles and facilitate m6A deposition (
      • Ping X.L.
      • Sun B.F.
      • Wang L.
      • Xiao W.
      • Yang X.
      • Wang W.J.
      • Adhikari S.
      • Shi Y.
      • Lv Y.
      • Chen Y.S.
      • Zhao X.
      • Li A.
      • Yang Y.
      • Dahal U.
      • Lou X.M.
      • et al.
      Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase.
      ). Virilizar was first found to be involved in sex determination in Drosophila (
      • Hilfiker A.
      • Nothiger R.
      The temperature-sensitive mutation vir ts (virilizer) identifies a new gene involved in sex determination of Drosophila.
      ). VIRMA, a mammal homolog of the Drosophila Virilizar, was found to interact with METTL3 via its N-terminus and characterized as a subunit of m6A methyltransferase complex (
      • Schwartz S.
      • Mumbach M.
      • Jovanovic M.
      • Wang T.
      • Maciag K.
      • Bushkin G.G.
      • Mertins P.
      • Ter-Ovanesyan D.
      • Habib N.
      • Cacchiarelli D.
      Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites.
      ). More recently, VIRMA was verified to associate with alternative polyadenylation via preferentially mediating m6A deposition on 3′ UTRs near the stop codon (
      • Yue Y.
      • Liu J.
      • Cui X.
      • Cao J.
      • Luo G.
      • Zhang Z.
      • Cheng T.
      • Gao M.
      • Shu X.
      • Ma H.
      • Wang F.
      • Wang X.X.
      • Shen B.
      • Wang Y.Z.
      • Feng X.H.
      • et al.
      VIRMA mediates preferential m6A mRNA methylation in 3′ UTR and near stop codon and associates with alternative polyadenylation.
      ).
      Before WTAP was identified as the subunit of mRNA m6A methyltransferase complex, an early co-immunoprecipitation study had indicated that WTAP can form a novel protein complex with VIRMA, HAKAI, RBM15, ZC3H13, Bcl-2-associated transcription factor 1 (BCLAF1), and thyroid hormone receptor-associated protein 3 (THRAP3) (
      • Horiuchi K.
      • Kawamura T.
      • Iwanari H.
      • Ohashi R.
      • Naito M.
      • Kodama T.
      • Hamakubo T.
      Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.
      ). Inspired by the result, VIRMA, HAKAI, RBM15, and ZC3H13 now have been characterized as subunits of mRNA m6A methyltransferase complex (
      • Schwartz S.
      • Mumbach M.
      • Jovanovic M.
      • Wang T.
      • Maciag K.
      • Bushkin G.G.
      • Mertins P.
      • Ter-Ovanesyan D.
      • Habib N.
      • Cacchiarelli D.
      Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites.
      ,
      • Růžička K.
      • Zhang M.
      • Campilho A.
      • Bodi Z.
      • Kashif M.
      • Saleh M.
      • Eeckhout D.
      • El-Showk S.
      • Li H.Y.
      • Zhong S.L.
      • Jaeger G.D.
      • Mongan N.P.
      • Hejátko J.
      • Helariutta Y.
      • Fray R.G.
      Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
      ,
      • Patil D.P.
      • Chen C.-K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • Jaffrey S.R.
      m6A RNA methylation promotes XIST-mediated transcriptional repression.
      ,
      • Wen J.
      • Lv R.
      • Ma H.
      • Shen H.
      • He C.
      • Wang J.
      • Jiao F.
      • Liu H.
      • Yang P.
      • Tan L.
      • Lan F.
      • Shi Y.J.
      • He C.
      • Shi Y.
      • Diao J.B.
      Zc3h13 regulates nuclear RNA m6A methylation and mouse embryonic stem cell self-renewal.
      ). HAKAI, an E3 ubiquitin ligase, was first identified as a subunit of m6A methyltransferase complex in Arabidopsis (
      • Růžička K.
      • Zhang M.
      • Campilho A.
      • Bodi Z.
      • Kashif M.
      • Saleh M.
      • Eeckhout D.
      • El-Showk S.
      • Li H.Y.
      • Zhong S.L.
      • Jaeger G.D.
      • Mongan N.P.
      • Hejátko J.
      • Helariutta Y.
      • Fray R.G.
      Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
      ). The knockdown of HAKAI in mammalian cells leads to a reduction of 23% in m6A abundance, while its disruption in Arabidopsis leads to a more moderate decrease in m6A levels with no obvious phenotypical consequence (
      • Růžička K.
      • Zhang M.
      • Campilho A.
      • Bodi Z.
      • Kashif M.
      • Saleh M.
      • Eeckhout D.
      • El-Showk S.
      • Li H.Y.
      • Zhong S.L.
      • Jaeger G.D.
      • Mongan N.P.
      • Hejátko J.
      • Helariutta Y.
      • Fray R.G.
      Identification of factors required for m6A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI.
      ). RNA-binding motif protein 15 (RBM15) as a subunit of m6A methyltransferase complex allows the formation of m6A in long noncoding RNA X-inactive specific transcript (XIST) and mRNA transcripts. The knockdown of RBM15 impairs XIST-mediated gene silencing in a manner that depends on YTH domain containing 1 (YTHDC1) (
      • Patil D.P.
      • Chen C.-K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • Jaffrey S.R.
      m6A RNA methylation promotes XIST-mediated transcriptional repression.
      ). The zinc-finger protein ZC3H13 plays an important role in regulating m6A formation in the nucleus. The knockdown of ZC3H13 results in the translocation of WTAP, HAKAI, and VIRMA to the cytoplasm, which dramatically reduces m6A level by 60 to 70% and affects the self-renewal of mESCs (
      • Wen J.
      • Lv R.
      • Ma H.
      • Shen H.
      • He C.
      • Wang J.
      • Jiao F.
      • Liu H.
      • Yang P.
      • Tan L.
      • Lan F.
      • Shi Y.J.
      • He C.
      • Shi Y.
      • Diao J.B.
      Zc3h13 regulates nuclear RNA m6A methylation and mouse embryonic stem cell self-renewal.
      ).

      Other m6A writers

      Apart from the mRNA m6A methyltransferase complex, several other methyltransferases are able to add m6A in other RNA types. For example, ZCCHC4 installs m6A at A4220 position of 28S rRNA, and knockdown of ZCCHC4 leads to the downregulation of translation and inhibits cell proliferation (
      • Ren W.D.
      • Lu J.W.
      • Huang M.J.
      • Gao L.F.
      • Li D.X.
      • Wang G.G.
      • Song J.K.
      Structure and regulation of ZCCHC4 in m6A-methylation of 28S rRNA.
      ). METTL5, an 18S rRNA m6A methyltransferase, can form a heterodimer with tRNA methyltransferase homolog 112 (TRMT112) to gain metabolic stability through the formation of a parallel β-zipper between main chain atoms (
      • Van Tran N.
      • Ernst F.G.M.
      • Hawley B.R.
      • Zorbas C.
      • Ulryck N.
      • Hackert P.
      • Bohnsack K.E.
      • Bohnsack M.T.
      • Jaffrey S.R.
      • Graille M.
      The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112.
      ). METTL16 is a U6 spliceosomal small nuclear RNA (snRNA) m6A methyltransferase and also specifically deposits m6A at an intron of SAM synthetase MAT2A that carries an evolutionarily conserved U6 m6A motif UACm6AGAGAA (
      • Pendleton K.E.
      • Chen B.
      • Liu K.Q.
      • Hunter O.V.
      • Xie Y.
      • Tu B.P.
      • Conrad N.K.
      The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention.
      ). The m6A on MAT2A pre-mRNA deposited by METTL16 leads to MTA2A intron retention and nuclear degradation, consequently regulating intracellular SAM homeostasis and mouse early embryonic development (
      • Pendleton K.E.
      • Chen B.
      • Liu K.Q.
      • Hunter O.V.
      • Xie Y.
      • Tu B.P.
      • Conrad N.K.
      The U6 snRNA m6A methyltransferase METTL16 regulates SAM synthetase intron retention.
      ,
      • Warda A.S.
      • Kretschmer J.
      • Hackert P.
      • Lenz C.
      • Urlaub H.
      • Höbartner C.
      • Sloan K.E.
      • Bohnsack M.T.
      Human METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs.
      ,
      • Shima H.
      • Matsumoto M.
      • Ishigami Y.
      • Ebina M.
      • Muto A.
      • Sato Y.
      • Kumagai S.
      • Ochiai K.
      • Suzuki T.
      • Igarashi K.
      S-Adenosylmethionine synthesis is regulated by selective N6-adenosine methylation and mRNA degradation involving METTL16 and YTHDC1.
      ). Although depletion of human METTL16 increases lots of m6A methylation sites in mRNA (
      • Warda A.S.
      • Kretschmer J.
      • Hackert P.
      • Lenz C.
      • Urlaub H.
      • Höbartner C.
      • Sloan K.E.
      • Bohnsack M.T.
      Human METTL16 is a N6-methyladenosine (m6A) methyltransferase that targets pre-mRNAs and various non-coding RNAs.
      ), recent finding revealed that the increased m6A sites lacking the UACm6AGAGAA motif were mediated by the reduced intercellular SAM level (
      • Shima H.
      • Matsumoto M.
      • Ishigami Y.
      • Ebina M.
      • Muto A.
      • Sato Y.
      • Kumagai S.
      • Ochiai K.
      • Suzuki T.
      • Igarashi K.
      S-Adenosylmethionine synthesis is regulated by selective N6-adenosine methylation and mRNA degradation involving METTL16 and YTHDC1.
      ), suggesting that mammalian METTL16 only directly methylates mRNA containing the UACm6AGAGAA motifs.

      m6A erasers

      Early genome-wide association studies have shown that FTO influences human obesity and energy homeostasis (
      • Dina C.
      • Meyre D.
      • Gallina S.
      • Durand E.
      • Körner A.
      • Jacobson P.
      • Carlsson L.M.
      • Kiess W.
      • Vatin V.
      • Lecoeur C.
      • Delplanque J.
      • Vaillant E.
      • Pattou F.
      • Ruiz J.
      • Weill J.
      • et al.
      Variation in FTO contributes to childhood obesity and severe adult obesity.
      ,
      • 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.
      A common variant in the FTO gene is associated with body mass index and predisposes to childhood and adult obesity.
      ,
      • Scuteri A.
      • Sanna S.
      • Chen W.-M.
      • Uda M.
      • Albai G.
      • Strait J.
      • Najjar S.
      • Nagaraja R.
      • Orrú M.
      • Usala G.
      • Dei M.
      • Lai S.
      • Maschio A.
      • Busonero F.
      • Mulas A.
      • et al.
      Genome-wide association scan shows genetic variants in the FTO gene are associated with obesity-related traits.
      ,
      • Scott L.J.
      • Mohlke K.L.
      • Bonnycastle L.L.
      • Willer C.J.
      • Li Y.
      • Duren W.L.
      • Erdos M.R.
      • Stringham H.M.
      • Chines P.S.
      • Jackson A.U.
      A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants.
      ). The overexpression of FTO in mice leads to an increased food intake and obesity, and the knockout of FTO in mice leads to significant reductions in body mass and growth retardation (
      • Boissel S.
      • Reish O.
      • Proulx K.
      • Kawagoe-Takaki H.
      • Sedgwick B.
      • Yeo G.S.
      • Meyre D.
      • Golzio C.
      • Molinari F.
      • Kadhom N.
      Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations.
      ,
      • Church C.
      • Moir L.
      • McMurray F.
      • Girard C.
      • Banks G.T.
      • Teboul L.
      • Wells S.
      • Brüning J.C.
      • Nolan P.M.
      • Ashcroft M.F.
      • Cox R.D.
      Overexpression of Fto leads to increased food intake and results in obesity.
      ,
      • Fischer J.
      • Koch L.
      • Emmerling C.
      • Vierkotten J.
      • Peters T.
      • Brüning J.C.
      • Rüther U.
      Inactivation of the Fto gene protects from obesity.
      ). Patients with loss-of-function mutation R316Q in FTO (R316Q mutation abolishes FTO enzyme function) have a severe polymalformation syndrome (
      • Boissel S.
      • Reish O.
      • Proulx K.
      • Kawagoe-Takaki H.
      • Sedgwick B.
      • Yeo G.S.
      • Meyre D.
      • Golzio C.
      • Molinari F.
      • Kadhom N.
      Loss-of-function mutation in the dioxygenase-encoding FTO gene causes severe growth retardation and multiple malformations.
      ), which shows that the demethylation activity of FTO is required for normal developments of central nervous and cardiovascular systems in human. FTO is initially regarded as a demethylase that oxidatively demethylates N3-methylthymidine (3 mT) in ssDNA and N3-methyluridine (m3U) in ssRNA (
      • Gerken T.
      • Girard C.A.
      • Tung Y.-C.L.
      • Webby C.J.
      • Saudek V.
      • Hewitson K.S.
      • Yeo G.S.
      • McDonough M.A.
      • Cunliffe S.
      • McNeill L.A.
      The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase.
      ,
      • Jia G.F.
      • Yang C.G.
      • Yang S.D.
      • Jian X.
      • Yi C.Q.
      • Zhou Z.Q.
      • He C.
      Oxidative demethylation of 3-methylthymine and 3-methyluracil in single-stranded DNA and RNA by mouse and human FTO.
      ). However, DNA modification 3 mT and RNA modification m3U are extremely rare in vivo, suggesting that these two modifications are not the physiological substrates of FTO for the striking phenotypes observed in FTO-deficient mice or patients (
      • Church C.
      • Moir L.
      • McMurray F.
      • Girard C.
      • Banks G.T.
      • Teboul L.
      • Wells S.
      • Brüning J.C.
      • Nolan P.M.
      • Ashcroft M.F.
      • Cox R.D.
      Overexpression of Fto leads to increased food intake and results in obesity.
      ,
      • Fischer J.
      • Koch L.
      • Emmerling C.
      • Vierkotten J.
      • Peters T.
      • Brüning J.C.
      • Rüther U.
      Inactivation of the Fto gene protects from obesity.
      ). Later, FTO was identified as an m6A demethylase in nuclear RNA (
      • Jia G.F.
      • Fu Y.
      • Zhao X.
      • Dai Q.
      • Zheng G.Q.
      • Yang Y.
      • Yi C.Q.
      • Lindahl T.
      • Pan T.
      • Yang Y.G.
      • He C.
      N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO.
      ), uncovering the reversibility of RNA modification. The m6A demethylation of FTO regulates adipogenesis through m6A-mediated alternative splicing of Runt-related transcription factor 1 (RUNX1T1), an adipogenesis-related transcription factor, which provides a novel mechanism explaining obesity (
      • Merkestein M.
      • Laber S.
      • McMurray F.
      • Andrew D.
      • Sachse G.
      • Sanderson J.
      • Li M.
      • Usher S.
      • Sellayah D.
      • Ashcroft F.M.
      • Cox R.D.
      FTO influences adipogenesis by regulating mitotic clonal expansion.
      ,
      • 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.
      FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis.
      ).
      FTO can also demethylate other RNA methylation modifications such as cap m6Am and m1A with varying efficiency and its subcellular localization affects the substrates specificity (
      • Mauer J.
      • Luo X.
      • Blanjoie A.
      • Jiao X.
      • Grozhik A.V.
      • Patil D.P.
      • Linder B.
      • Pickering B.F.
      • Vasseur J.-J.
      • Chen Q.
      • Gross S.S.
      • Elemento O.
      • Debart F.
      • Kiledjian M.
      • Jaffrey S.R.
      Reversible methylation of m6Am in the 5′ cap controls mRNA stability.
      ,
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • Shi H.
      • Cui X.
      • Su R.
      • Klungland A.
      • Jia G.F.
      • Chen J.J.
      • He C.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
      ,
      • Mauer J.
      • Sindelar M.
      • Despic V.
      • Guez T.
      • Hawley B.R.
      • Vasseur J.-J.
      • Rentmeister A.
      • Gross S.S.
      • Pellizzoni L.
      • Debart F.
      • Goodarzi H.
      • Jaffrey R.S.
      FTO controls reversible m6Am RNA methylation during snRNA biogenesis.
      ). In the nucleus, FTO preferentially demethylates internal m6A in poly(A) RNA, m6A and m6Am in snRNA, and m1A in tRNA; in the cytoplasm, FTO demethylates both internal m6A and cap m6Am in poly(A) and m1A in tRNA (
      • Wei J.
      • Liu F.
      • Lu Z.
      • Fei Q.
      • Ai Y.
      • He P.C.
      • Shi H.
      • Cui X.
      • Su R.
      • Klungland A.
      • Jia G.F.
      • Chen J.J.
      • He C.
      Differential m6A, m6Am, and m1A demethylation mediated by FTO in the cell nucleus and cytoplasm.
      ). The crystal structure of FTO bound to 6mA-modified ssDNA reveals the molecular basis of the recognition and catalytic demethylation of FTO toward different substrates (
      • Zhang X.
      • Wei L.-H.
      • Wang Y.
      • Xiao Y.
      • Liu J.
      • Zhang W.
      • Yan N.
      • Amu G.
      • Tang X.
      • Zhang L.
      • Jia G.F.
      Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates.
      ) (Fig. 2C), demonstrating that N6-methyladenine is the most favorable nucleobase substrate of FTO. FTO uses the ligands R96 and E234 to form three H bonds with the N1, N6, and N7 atoms of the 6 mA purine ring for locking the 6 mA base in the catalytic pocket and utilizes the side chains of R96, Y106, Y108, L203, and R322 ligands to form a hydrophobic pocket for stabilization of the N6-methyl group at the orientation of Fe(II) and α-ketoglutarate (α-KG) for oxidation (Fig. 2C). Compared with the recognition of FTO toward m6A, the weak H bond between the amide nitrogen of E234 and O4 atom of 3 mT and the loss of H bond between the purine ring of m1A with both E234 and R96 may explain why FTO exhibits slower enzymatic activity for 3 mT and m1A than for m6A (
      • Zhang X.
      • Wei L.-H.
      • Wang Y.
      • Xiao Y.
      • Liu J.
      • Zhang W.
      • Yan N.
      • Amu G.
      • Tang X.
      • Zhang L.
      • Jia G.F.
      Structural insights into FTO’s catalytic mechanism for the demethylation of multiple RNA substrates.
      ,
      • Han Z.
      • Niu T.
      • Chang J.
      • Lei X.
      • Zhao M.
      • Wang Q.
      • Cheng W.
      • Wang J.
      • Feng Y.
      • Chai J.
      Crystal structure of the FTO protein reveals basis for its substrate specificity.
      ).
      Splicing factor proline and glutamine-rich (SFPQ) is a multifunctional nuclear protein that participates in several cellular activities, including RNA transport, apoptosis, and DNA repair (
      • Pellarin I.
      • Dall’Acqua A.
      • Gambelli A.
      • Pellizzari I.
      • D’Andrea S.
      • Sonego M.
      • Lorenzon I.
      • Schiappacassi M.
      • Belletti B.
      • Baldassarre G.
      Splicing factor proline-and glutamine-rich (SFPQ) protein regulates platinum response in ovarian cancer-modulating SRSF2 activity.
      ). Via the genetically encoded and site-specific photo-cross-linking strategy, SFPQ is identified to directly interact with FTO and to facilitate its choice of specific RNAs for FTO-mediated m6A demethylation (
      • Song H.
      • Wang Y.
      • Wang R.
      • Zhang X.
      • Liu Y.
      • Jia G.
      • Chen P.R.
      SFPQ is an FTO-binding protein that facilitates the demethylation substrate preference.
      ), which implies the possibility that protein–protein interactions are another regulatory factor affecting FTO’s substrates preference.
      ALKBH5 is the second identified m6A demethylase that oxidatively demethylates m6A in mRNA both in vitro and in vivo (
      • Zheng G.Q.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • Vågbø C.B.
      • Shi Y.
      • Wang W.L.
      • Song S.H.
      • Lu Z.
      • Bosmans R.P.G.
      • Dai Q.
      • Hao Y.J.
      • Yang X.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      ). Similar to METTL3, ALKBH5 colocalizes with nuclear speckles and influences mRNA processing, which ultimately affects mRNA export and metabolism (
      • Zheng G.Q.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • Vågbø C.B.
      • Shi Y.
      • Wang W.L.
      • Song S.H.
      • Lu Z.
      • Bosmans R.P.G.
      • Dai Q.
      • Hao Y.J.
      • Yang X.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      ,
      • Tang C.
      • Klukovich R.
      • Peng H.
      • Wang Z.
      • Yu T.
      • Zhang Y.
      • Zheng H.
      • Klungland A.
      • Yan W.
      ALKBH5-dependent m6A demethylation controls splicing and stability of long 3′-UTR mRNAs in male germ cells.
      ). The knockdown or overexpression of ALKBH5 can increase or decrease mRNA m6A abundance, respectively. The ALKBH5-deficient male mice exhibit compromised spermatogenesis (
      • Zheng G.Q.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • Vågbø C.B.
      • Shi Y.
      • Wang W.L.
      • Song S.H.
      • Lu Z.
      • Bosmans R.P.G.
      • Dai Q.
      • Hao Y.J.
      • Yang X.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      ). Unlike FTO, ALKBH5 demethylases m6A highly specifically with no other substrates found so far.
      Although ALKBH5 and FTO both belong to the Fe(II)/α-KG-dependent dioxygenase AlkB family, their catalytical mechanisms for m6A demethylation are different. FTO performs twice oxidation reactions to oxidize m6A to N6-hydroxymethyladenosine (hm6A) quickly and to further oxidize hm6A to N6-formyladenosine (f6A) slowly. The intermediates hm6A and f6A are not stable (∼3 h at neutral pH) and automatically form adenosine by releasing formaldehyde and formic acid, respectively (
      • Fu Y.
      • Jia G.F.
      • Pang X.Q.
      • Wang R.N.
      • Wang X.
      • Li C.J.
      • Smemo S.
      • Dai Q.
      • Bailey K.A.
      • Nobrega M.A.
      • Han K.L.
      • Cui Q.
      • He C.
      FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
      ) (Fig. 2D). Distinct from FTO, ALKBH5 catalyzes a direct m6A-to-A transformation with rapid formaldehyde release (
      • Fu Y.
      • Jia G.F.
      • Pang X.Q.
      • Wang R.N.
      • Wang X.
      • Li C.J.
      • Smemo S.
      • Dai Q.
      • Bailey K.A.
      • Nobrega M.A.
      • Han K.L.
      • Cui Q.
      • He C.
      FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
      ,
      • Toh J.D.
      • Crossley S.W.
      • Bruemmer K.J.
      • Eva J.G.
      • He D.
      • Iovan D.A.
      • Chang C.J.
      Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.
      ) (Fig. 2D). Specifically, the hydrogen bond between R96 of FTO and N1 atom of purine ring imbues the nucleobase with a partial positive charge and inductively and mesomerically lowers the highest-occupied molecular orbital of the N6 lone pair, thereby disfavoring Schiff base formation and forming hm6A as the product. However, K132 and Y139 of ALKBH5 in the nucleobase recognition region facilitate the formation of a covalent intermediate via elimination of H2O (
      • Toh J.D.
      • Crossley S.W.
      • Bruemmer K.J.
      • Eva J.G.
      • He D.
      • Iovan D.A.
      • Chang C.J.
      Distinct RNA N-demethylation pathways catalyzed by nonheme iron ALKBH5 and FTO enzymes enable regulation of formaldehyde release rates.
      ) (Fig. 2E). The proposed mechanism could explain the difference of catalytical oxidation between FTO and ALKBH5. However, it is still unclear about the catalytical oxidation mechanism how FTO recognizes hm6A to perform the second round oxidation.

      The applications of m6A writers and erasers in m6A detection

      It is of prime importance to identify the amount and distribution of m6A in RNA molecules in order to understand its biological functions. The development of mass spectrometric techniques and high-throughput sequencing has facilitated the successive development of novel m6A detection approaches, providing useful and valuable tools for studying the physiological functions of m6A (
      • Jora M.
      • Lobue P.A.
      • Ross R.L.
      • Williams B.
      • Addepalli B.
      Detection of ribonucleoside modifications by liquid chromatography coupled with mass spectrometry.
      ,
      • Reuter J.A.
      • Spacek D.V.
      • Snyder M.P.
      High-throughput sequencing technologies.
      ). m6A antibody-based m6A sequencing (MeRIP) as the first generation of m6A sequencing method provides the transcriptome-wide m6A distribution information and quickens the functional study of m6A (
      • Dominissini D.
      • Moshitch-Moshkovitz S.
      • Schwartz S.
      • Salmon-Divon M.
      • Ungar L.
      • Osenberg S.
      • Cesarkas K.
      • Jacob-Hirsch J.
      • Amariglio N.
      • Kupiec M.
      • Sorek R.
      • Rechavi G.
      Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
      ,
      • 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.
      ). However, MeRIP method relies on the specificity of m6A antibody, and its resolution is quite low (at least 200 nt) (
      • McIntyre A.B.
      • Gokhale N.S.
      • Cerchietti L.
      • Jaffrey S.R.
      • Horner S.M.
      • Mason C.E.
      Limits in the detection of m6A changes using MeRIP/m6A-seq.
      ). To overcome these shortcomings, more advanced methods are urgently required to study the role of m6A in living organisms. In recent years, with the in-depth study of the enzymatic mechanisms of m6A writers and erasers, they have been applied to develop new m6A detection methods (
      • Zhang Z.
      • Chen L.Q.
      • Zhao Y.L.
      • Yang C.G.
      • Roundtree I.A.
      • Zhang Z.J.
      • Ren J.
      • Xie W.
      • He C.
      • Luo G.Z.
      Single-base mapping of m6A by an antibody-independent method.
      ,
      • Garcia-Campos M.A.
      • Edelheit S.
      • Toth U.
      • Safra M.
      • Shachar R.
      • Viukov S.
      • Winkler R.
      • Nir R.
      • Lasman L.
      • Brandis A.
      Deciphering the “m6A code” via antibody-independent quantitative profiling.
      ,
      • Shu X.
      • Cao J.
      • Cheng M.H.
      • Xiang S.Y.
      • Gao M.S.
      • Li T.
      • Ying X.E.
      • Wang F.Q.
      • Yue Y.N.
      • Lu Z.K.
      • Dai Q.
      • Cui X.L.
      • Ma L.J.
      • Wang Y.Z.
      • He C.
      • et al.
      A metabolic labeling method detects m6A transcriptome-wide at single base resolution.
      ,
      • Wang Y.
      • Xiao Y.
      • Dong S.
      • Yu Q.
      • Jia G.
      Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
      ,
      • Xiao Y.
      • Wang Y.
      • Tang Q.
      • Wei L.H.
      • Zhang X.
      • Jia G.F.
      An elongation-and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N6-methyladenosine modification.
      ) (Fig. 3).
      Figure thumbnail gr3
      Figure 3The workflow of m6A detection methods assisted by m6A writers or erasers. A, MeRIP-seq/miCLIP/PA-m6A-seq, immunoprecipitation with m6A-specific antibody. B, MAZTER-seq/m6A-REF-seq, FTO-assisted differential digestion toward m6A and A. C, m6A-label-seq, metabolic labeling with N6-allyladenosine through methyltransferase as m6A putative sites. D, m6A-SEAL-seq, immunoprecipitation with biotin labeling to hm6A produced by FTO-assisted oxidation. E, SELECT, FTO assisted single-base elongation and ligation with PCR amplification for quantification.

      Antibody-based m6A sequencing methods

      The most common strategy for transcriptome-wide detection of RNA modifications is RNA immunoprecipitation (RIP) with specific antibodies of RNA modifications coupled with high-throughput sequencing. m6A-seq (also termed MeRIP-seq) utilizes commercial anti-m6A antibody to incubate with fragmented mRNA and enriches the m6A-modified fragments for high-throughput sequencing (
      • Dominissini D.
      • Moshitch-Moshkovitz S.
      • Schwartz S.
      • Salmon-Divon M.
      • Ungar L.
      • Osenberg S.
      • Cesarkas K.
      • Jacob-Hirsch J.
      • Amariglio N.
      • Kupiec M.
      • Sorek R.
      • Rechavi G.
      Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
      ,
      • 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.
      ) (Fig. 3A). In this way, m6A-seq or MeRIP-seq provides the information of both m6A-modified genes and m6A locations with a resolution around 200 nt.
      In order to refine m6A locus more precisely, inspired by PAR-CLIP and iCLIP methods (
      • Huppertz I.
      • Attig J.
      • D’Ambrogio A.
      • Easton L.E.
      • Sibley C.R.
      • Sugimoto Y.
      • Tajnik M.
      • König J.
      • Ule J.
      iCLIP: Protein–RNA interactions at nucleotide resolution.
      ,
      • Hafner M.
      • Landthaler M.
      • Burger L.
      • Khorshid M.
      • Hausser J.
      • Berninger P.
      • Rothballer A.
      • Ascano M.
      • Jungkamp A.-C.
      • Munschauer M.
      PAR-CliP-a method to identify transcriptome-wide the binding sites of RNA binding proteins.
      ), m6A-seq was modified into two similar methods: photo-cross-linking-assisted m6A sequencing strategy (PA-m6A-seq) and m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) (
      • Chen K.
      • Lu Z.K.
      • Wang X.
      • Fu Y.
      • Luo G.Z.
      • Liu N.
      • Han D.L.
      • Dominissini D.
      • Dai Q.
      • Pan T.
      High-resolution N6-methyladenosine (m6A) map using photo-crosslinking-assisted m6A sequencing.
      ,
      • 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.
      ). PA-m6A-seq metabolically incorporates 4-thiouridine (4SU) into mRNA and covalently cross-links 4SU with nearby aromatic amino acid residues of m6A antibody upon 365 nm UV irradiation (
      • Chen K.
      • Lu Z.K.
      • Wang X.
      • Fu Y.
      • Luo G.Z.
      • Liu N.
      • Han D.L.
      • Dominissini D.
      • Dai Q.
      • Pan T.
      High-resolution N6-methyladenosine (m6A) map using photo-crosslinking-assisted m6A sequencing.
      ). This technique vastly improves m6A resolution up to around 23 nt, but can only be used in cells due to the requirement of 4SU metabolism. miCLIP exploits 254 nm irradiation to cross-link mRNA with m6A antibody and introduces nucleotide mismatches or truncation signatures near m6A sites during reverse transcription (
      • 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.
      ). Although miCLIP successfully achieves a desired single-base resolution of m6A mapping at a transcriptome-wide level, the number of identified m6A sites by miCLIP is limited due to the low cross-linking efficiency.
      Although the aforementioned m6A antibody-based m6A sequencing methods are widely applied, these methods have some unavoidable drawbacks. Commercial m6A antibodies from different resources show differences in affinity toward m6A, and m6A antibodies can also recognize non-m6A specific sequences and m6Am, which leads to undesirably high false-positive rates (
      • 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.
      ). Furthermore, the low reproducibility (30%–60%) was observed across MeRIP-seq datasets in the same cell types or even between biological replicates (
      • Chen K.
      • Lu Z.K.
      • Wang X.
      • Fu Y.
      • Luo G.Z.
      • Liu N.
      • Han D.L.
      • Dominissini D.
      • Dai Q.
      • Pan T.
      High-resolution N6-methyladenosine (m6A) map using photo-crosslinking-assisted m6A sequencing.
      ,
      • McIntyre A.B.
      • Gokhale N.S.
      • Cerchietti L.
      • Jaffrey S.R.
      • Horner S.M.
      • Mason C.E.
      Limits in the detection of m6A changes using MeRIP/m6A-seq.
      ). Together, it is extremely necessary and urgently needed to develop more accurate and precise m6A detection methods.

      m6A writer or eraser-based m6A detection methods

      m6A antibody-based profiling has displayed moderate effectiveness but with limitations in degree of accuracy and precision. m6A writers and erasers can directly and highly specifically interact with m6A, which can be used for overcoming the shortcomings rising from antibody-based methods.

      m6A-REF-seq and MAZTER-seq

      MazF is an m6A-sensitive endoribonuclease that cuts the ACA motif but not the m6ACA sequence (
      • Imanishi M.
      • Tsuji S.
      • Suda A.
      • Futaki S.
      Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease.
      ). Taken of this feature, two similar methods, MAZTER-seq and m6A-REF-seq, have been developed (
      • Zhang Z.
      • Chen L.Q.
      • Zhao Y.L.
      • Yang C.G.
      • Roundtree I.A.
      • Zhang Z.J.
      • Ren J.
      • Xie W.
      • He C.
      • Luo G.Z.
      Single-base mapping of m6A by an antibody-independent method.
      ,
      • Garcia-Campos M.A.
      • Edelheit S.
      • Toth U.
      • Safra M.
      • Shachar R.
      • Viukov S.
      • Winkler R.
      • Nir R.
      • Lasman L.
      • Brandis A.
      Deciphering the “m6A code” via antibody-independent quantitative profiling.
      ), which respectively exploit MazF to parallelly digest mRNAs isolated from control versus m6A writer knockout cells or RNA treated with FTO demethylation reaction versus without FTO treatment (Fig. 3B). The m6A removal by either in vivo knockout of m6A methyltransferase or in vitro FTO demethylation reaction becomes the key step to determine the specific site of m6A by comparing the cutting reads at m6A sites between paired samples. Both methods provide highly accurate and precise information of m6A distribution at single-base resolution; however, they can only identify ∼16 to 25% m6A sites because of the restrictions of MazF specifically recognizing ACA motif.

      m6A-label-seq

      Similar to other methylation enzymes, the methyl group donor SAM acts as a cosubstrate of the m6A methyltransferase complex and is responsible for transferring a methyl group to the N6-position of specific adenosines. A metabolic labeling method was developed to detect mRNA m6A from transcriptome-wide range at single-base resolution (m6A-label-seq) (
      • Shu X.
      • Cao J.
      • Cheng M.H.
      • Xiang S.Y.
      • Gao M.S.
      • Li T.
      • Ying X.E.
      • Wang F.Q.
      • Yue Y.N.
      • Lu Z.K.
      • Dai Q.
      • Cui X.L.
      • Ma L.J.
      • Wang Y.Z.
      • He C.
      • et al.
      A metabolic labeling method detects m6A transcriptome-wide at single base resolution.
      ). This approach metabolically modifies adenosine into N6-allyladenosine (a6A) at the supposed m6A-generating sites by leveraging Se-allyl-l-selenohomocysteine, which forms allyl-SAM (substituting the methyl group on SAM with an allyl) in cells. The produced a6A modifications in mRNA are enriched by antibody and further performed iodination-induced cyclization. The cyclized a6A can induce base misincorporation during reverse transcription and thus provides the transcriptome-wide m6A location at single-base resolution (Fig. 3C). However, m6A-label-seq requires the metabolism of Se-allyl-l-selenohomocysteine and only can be used in cellular system.

      m6A-SEAL-seq

      FTO can oxidize m6A twice to generate hm6A and f6A as intermediate modifications with a half-life of ∼3 h in an aqueous solution under physiological relevant conditions (
      • Fu Y.
      • Jia G.F.
      • Pang X.Q.
      • Wang R.N.
      • Wang X.
      • Li C.J.
      • Smemo S.
      • Dai Q.
      • Bailey K.A.
      • Nobrega M.A.
      • Han K.L.
      • Cui Q.
      • He C.
      FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
      ). Recently, a FTO-assisted m6A selective chemical labeling method, termed m6A-SEAL-seq, utilizes a thiol addition reaction to selectively label the m6A oxidation production, hm6A, for m6A detection (
      • Wang Y.
      • Xiao Y.
      • Dong S.
      • Yu Q.
      • Jia G.
      Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
      ). In this process, m6A modification in fragmented mRNA is first oxidized by FTO into an unstable hm6A, which further reacts with dithiothreitol (DTT) to form stable N6-dithiolsitolmethyladenosine (dm6A). The free sulfhydryl group on dm6A can be biotinylated for enrichment and sequencing subsequently. m6A-SEAL-seq is an antibody-free and chemically covalent cross-linking method for m6A detection with ∼200 nt resolution (Fig. 3D). The validation of m6A-SEAL identified m6A sites revealed that m6A-SEAL-seq has a greater specificity and sensitivity than other available methods, but it needs to be improved to single-base resolution in the future.

      SELECT

      It is highly desirable to develop m6A locus detection method at the single gene level in order to perform studies of m6A biological functions. Site-specific cleavage and radioactive labeling of the modified nucleotides followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) are some of the most reliable techniques to detect the presence and modification fraction of m6A (
      • Liu N.
      • Parisien M.
      • Dai Q.
      • Zheng G.
      • He C.
      • Pan T.
      Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA.
      ). Briefly, the RNase H cleaves the 5′ of the specific site in a candidate sequence by a complementary 2′-OMe/2′-H chimeric oligonucleotide, followed by radiolabeling with 32P and splinted-ligation of ssDNA. After digesting with RNase T1/A and nuclease, it is possible to distinguish and quantify P1, 32P-A, and 32P-m6A through thin-layer chromatography to indicate the m6A modification status at the candidate sites. SCARLET is the first method to achieve a precise determination of the location and modification proportion of m6A in mRNA or lncRNA. However, it should be noted that the time-consuming and radiolabeling properties make this approach difficult and far from ideal for extensive applications.
      A single-base elongation- and ligation-based qPCR amplification method (SELECT) can perfectly quantify m6A locus and fraction at a specific site in mRNA and lncRNA (
      • Xiao Y.
      • Wang Y.
      • Tang Q.
      • Wei L.H.
      • Zhang X.
      • Jia G.F.
      An elongation-and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N6-methyladenosine modification.
      ) (Fig. 3E), which is easy, convenient, and free of radiolabeling. Specifically, SELECT takes advantage of the fact that m6A blocks the single-base elongation activity of DNA polymerase and the nick ligation efficiency of ligases, which influence the formation of intact complementary DNA targeting candidate RNA sequences and ultimately result in a discrepant number of qPCR cycles between m6A and non-m6A samples. Assisted by FTO-mediated m6A demethylation, SELECT can easily distinguish m6A sites in mRNA or lncRNA through comparing the qPCR cycles between total RNA treated with FTO demethylation reaction and without FTO treatment.
      The newly developed m6A writers- and erasers-assisted m6A detection methods should be further improved. MAZTER-seq and m6A-REF-seq lose most of the m6A sites (
      • Zhang Z.
      • Chen L.Q.
      • Zhao Y.L.
      • Yang C.G.
      • Roundtree I.A.
      • Zhang Z.J.
      • Ren J.
      • Xie W.
      • He C.
      • Luo G.Z.
      Single-base mapping of m6A by an antibody-independent method.
      ,
      • Garcia-Campos M.A.
      • Edelheit S.
      • Toth U.
      • Safra M.
      • Shachar R.
      • Viukov S.
      • Winkler R.
      • Nir R.
      • Lasman L.
      • Brandis A.
      Deciphering the “m6A code” via antibody-independent quantitative profiling.
      ), which urges us to find new endoribonuclease capable of recognizing more universal sequence motifs while retaining the m6A sensitivity. In m6A-label-seq, the low labeling yield and moderate cellular stress induced by methionine analog result in the loss of endogenous m6A sites and the change of nascent RNA methylation status (
      • Shu X.
      • Cao J.
      • Cheng M.H.
      • Xiang S.Y.
      • Gao M.S.
      • Li T.
      • Ying X.E.
      • Wang F.Q.
      • Yue Y.N.
      • Lu Z.K.
      • Dai Q.
      • Cui X.L.
      • Ma L.J.
      • Wang Y.Z.
      • He C.
      • et al.
      A metabolic labeling method detects m6A transcriptome-wide at single base resolution.
      ). Currently it can only be used in cells due to the requirement of metabolism of methionine analog, and seeking a methyltransferase that can transfer the allyl group onto m6A sites in vitro would provide a good strategy for m6A sequencing. The introduction of DTT into methyl group on N6 position in m6A-SEAL-seq has no obvious effect on base paring between adenosine and thymine, and thus no mutation or stop signal occurs during reverse transcription, which makes it fail to provide precise distribution of m6A (
      • Wang Y.
      • Xiao Y.
      • Dong S.
      • Yu Q.
      • Jia G.
      Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
      ). m6A-SEAL-seq needs to be further modified to introduce mismatches or truncations during reverse transcription for achieving the single-base resolution.

      The functions of m6A based on m6A writers and erasers

      m6A modifications have shown significant effects on many aspects of physiological processes including regulating the circadian rhythm, spermatogenesis, stem cell differentiation, viral infection, cancer progression, and so on (
      • Batista P.J.
      • Molinie B.
      • Wang J.
      • Qu K.
      • Zhang J.
      • Li L.
      • Bouley D.M.
      • Lujan E.
      • Haddad B.
      • Daneshvar K.
      m6A RNA modification controls cell fate transition in mammalian embryonic stem cells.
      ,
      • Hastings M.H.
      m6A mRNA methylation: A new circadian pacesetter.
      ,
      • Lin Z.
      • Hsu P.J.
      • Xing X.D.
      • Fang J.H.
      • Lu Z.K.
      • Zou Q.
      • Zhang K.J.
      • Zhang X.
      • Zhou Y.C.
      • Zhang T.
      • Zhang Y.C.
      • Song W.L.
      • Jia G.F.
      • Yang X.R.
      • He C.
      • et al.
      Mettl3-/Mettl14-mediated mRNA N6-methyladenosine modulates murine spermatogenesis.
      ,
      • Williams G.D.
      • Gokhale N.S.
      • Horner S.M.
      Regulation of viral infection by the RNA modification N6-methyladenosine.
      ,
      • Lan Q.
      • Liu P.Y.
      • Haase J.
      • Bell J.L.
      • Hüttelmaier S.
      • Liu T.
      The critical role of RNA m6A methylation in cancer.
      ). Different m6A readers bind m6A modifications to regulate mRNA processing and metabolism (
      • Wang X.
      • Zhao B.X.S.
      • Roundtree I.A.
      • Lu Z.K.
      • Han D.L.
      • Ma H.H.
      • Weng X.C.
      • Chen K.
      • Shi H.L.
      • He C.
      N6-methyladenosine modulates messenger RNA translation efficiency.
      ,
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • Fu Y.
      • Parisien M.
      • Dai Q.
      • Jia G.
      • Ren B.
      • Pan T.
      • He C.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Zhang Z.
      • Theler D.
      • Kaminska K.H.
      • Hiller M.
      • Stamm S.
      The YTH domain is a novel RNA binding domain.
      ,
      • Xu C.
      • Wang X.
      • Liu K.
      • Roundtree A.I.
      • Tempel W.
      • Li Y.J.
      • Lu Z.K.
      • He C.
      • Min J.R.
      Structural basis for selective binding of m6A RNA by the YTHDC1 YTH domain.
      ,
      • Alarcón C.R.
      • Goodarzi H.
      • Lee H.
      • Liu X.
      • Tavazoie S.
      • Tavazoie S.F.
      HNRNPA2B1 is a mediator of m6A-dependent nuclear RNA processing events.
      ,
      • Liu M.
      • Dai Q.
      • Zheng G.
      • He C.
      • Parisien M.
      • Pan T.
      N6-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions.
      ,
      • 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.
      • Wang X.
      • Ma H.L.
      • Huang C.M.
      • Yang Y.
      • Huang N.
      • et al.
      Nuclear m6A reader YTHDC1 regulates mRNA splicing.
      ). The METTL3-METTL14-core complex and FTO/ALKBH5 have decisive impacts on m6A deposition and are thus crucial for m6A-dependent biological functions. The aberrant expression of writers and erasers influences a series of physiological activities (
      • Peng W.
      • Li J.
      • Chen R.
      • Gu Q.
      • Yang P.
      • Qian W.
      • Ji D.
      • Wang Q.
      • Zhang Z.
      • Tang J.
      Upregulated METTL3 promotes metastasis of colorectal cancer via miR-1246/SPRED2/MAPK signaling pathway.
      ,
      • Ma J.Z.
      • Yang F.
      • Zhou C.C.
      • Liu F.
      • Yuan J.H.
      • Wang F.
      • Wang T.T.
      • Xu Q.G.
      • Zhou W.P.
      • Sun S.H.
      METTL14 suppresses the metastatic potential of hepatocellular carcinoma by modulating N6-methyladenosine-dependent primary microRNA processing.
      ,
      • Zhang J.
      • Guo S.
      • Piao H.-Y.
      • Wang Y.
      • Wu Y.
      • Meng X.-Y.
      • Yang D.
      • Zheng Z.-C.
      • Zhao Y.
      ALKBH5 promotes invasion and metastasis of gastric cancer by decreasing methylation of the lncRNA NEAT1.
      ,
      • Shen F.
      • Huang W.
      • Huang J.-T.
      • Xiong J.
      • Yang Y.
      • Wu K.
      • Jia G.-F.
      • Chen J.
      • Feng Y.-Q.
      • Yuan B.-F.
      Decreased N6-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5.
      ). Here we focus on the functions of m6A writers and erasers in two major biological events, viral infection, and cancer progression, in which m6A were reported to play vital roles and the biological functions of m6A have undergone widespread and elaborate investigations, and we also introduce newly developed m6A writers- and erasers-assisted techniques for deeply and rigorously studying and verifying the physiological functions of m6A.

      Viral infection

      m6A multidirectionally influences the interactions between RNA virus and host. The host m6A writers and erasers determine the deposition of m6A in viral RNA and affect viral RNA replication and virus proliferation. In host cells, m6A on transcripts related to antiviral pathways also affects the host’s antiviral ability (
      • Dang W.
      • Xie Y.
      • Cao P.F.
      • Xin S.Y.
      • Wang J.
      • Li S.
      • Li Y.L.
      • Lu J.H.
      N6-methyladenosine and viral infection.
      ). (Fig. 4A).
      Figure thumbnail gr4
      Figure 4The function research of m6A modification by m6A writers and erasers. m6A writers and erasers play crucial roles in various diseases. A, in viral infection, m6A writers and erasers can regulate the m6A level of both viral RNA itself and host antiviral RNAs, affecting viral RNA replication and viral particle production. B, in cancers, m6A writers or eraser can act on the mRNA of oncogenes or tumor suppressor genes, thereby affecting the expression of related genes and tumorigenesis. C, function research of m6A modification via site-specific regulation of m6A by CRISPR/Cas conjugated with m6A writers or erasers. The main components of m6A editing system include m6A writers or erasers, linker, programmable RNA-binding proteins such as dCas9 or dCas13, and sgRNA. When fused with dCas9 or dCas13, m6A writers or erasers can edit specific A or m6A site under the guidance of sgRNA to realize the conversion between A and m6A.
      Although there is no reported m6A machinery gene in virus genomic RNA (gRNA), viral RNA has been known to contain m6A modification since the 1970s (
      • Lavi S.
      • Shatkin A.J.
      Methylated simian virus 40-specific RNA from nuclei and cytoplasm of infected BSC-1 cells.
      ,
      • Krug R.M.
      • Morgan M.A.
      • Shatkin A.J.
      Influenza viral mRNA contains internal N6-methyladenosine and 5'-terminal 7-methylguanosine in cap structures.
      ,
      • Sommer S.
      • Salditt-Georgieff M.
      • Bachenheimer S.
      • Darnell J.
      • Furuichi Y.
      • Morgan M.
      • Shatkin A.
      The methylation of adenovirus-specific nuclear and cytoplasmic RNA.
      ,
      • Dimock K.
      • Stoltzfus C.M.
      Sequence specificity of internal methylation in B77 avian sarcoma virus RNA subunits.
      ). m6A is a conserved regulatory modification in viral RNA across the flaviviridae family, whose RNA replicates exclusively in the cytoplasm (
      • Wengler G.
      • Wengler G.
      The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity.
      ). The host m6A writers and erasers are responsible for the installation and removal of m6A in virus RNA, and the host m6A readers recognize m6A of virus RNA for regulation of viral RNA metabolism. Both Hepatitis C virus (HCV) and Zika virus (ZIKV) contain m6A modifications and are manipulated by host m6A machinery (m6A writer, eraser, and reader) (
      • Gokhale N.S.
      • McIntyre A.B.
      • McFadden M.J.
      • Roder A.E.
      • Kennedy E.M.
      • Gandara J.A.
      • Hopcraft S.E.
      • Quicke K.M.
      • Vazquez C.
      • Willer J.
      N6-methyladenosine in Flaviviridae viral RNA genomes regulates infection.
      ,
      • Lichinchi G.
      • Zhao B.X.S.
      • Wu Y.
      • Lu Z.K.
      • Qin Y.
      • He C.
      • Rana T.M.
      Dynamics of human and viral RNA methylation during Zika virus infection.
      ) (Fig. 4A). Knockdown of host m6A writers or erasers can respectively promote or inhibit infectious particle production of HCV and ZIKV during viral infection, indicating m6A negatively regulates ZIKV or HCV infection. Further studies reveal that host m6A reader proteins YTHDF1-3 are able to relocalize at lipid droplets, viral assembly sites, under HCV infection and bind HCV RNAs to reduce HCV particle production (
      • Gokhale N.S.
      • McIntyre A.B.
      • McFadden M.J.
      • Roder A.E.
      • Kennedy E.M.
      • Gandara J.A.
      • Hopcraft S.E.
      • Quicke K.M.
      • Vazquez C.
      • Willer J.
      N6-methyladenosine in Flaviviridae viral RNA genomes regulates infection.
      ). Differently, the functions of m6A in ZIKV particle assembly occurs via YTHDF-mediated ZIKV RNA degradation (Fig. 4A).
      In HIV-1, 14 m6A methylation peaks have been identified in its coding and noncoding regions of HIV-1 gRNA, and 56 human transcripts are specifically methylated in HIV-1-infected T cells. Knockdown of host m6A writers or erasers decreases or increases HIV-1 replication by affecting the m6A methylation of HIV-1 Rev response element (RRE) RNA and the subsequent export of viral RNA. The m6A in HIV-1 RRE RNA enhances the binding of HIV-1 Rev and facilities the nuclear export of HIV-1 RNA (
      • Lichinchi G.
      • Gao S.
      • Saletore Y.
      • Gonzalez G.M.
      • Bansal V.
      • Wang Y.
      • Mason C.E.
      • Rana T.M.
      Dynamics of the human and viral m6A RNA methylomes during HIV-1 infection of T cells.
      ). Other studies reveal that in a postentry step of HIV-1 infection, host m6A writers can install m6A in HIV-1 RNAs in the cytoplasm and, subsequently, host YTHDF1-3 bind m6A-modified HIV-1 genomic RNA (gRNA), which reduces reverse transcription of HIV-1 gRNA into proviral dsDNA (
      • Kennedy E.M.
      • Bogerd H.P.
      • Kornepati A.V.
      • Kang D.
      • Ghoshal D.
      • Marshall J.B.
      • Poling B.C.
      • Tsai K.
      • Gokhale N.S.
      • Horner S.M.
      Posttranscriptional m6A editing of HIV-1 mRNAs enhances viral gene expression.
      ,
      • Lu W.
      • Tirumuru N.
      • Gelais C.S.
      • Koneru P.C.
      • Liu C.
      • Kvaratskhelia M.
      • He C.
      • Wu L.
      N6-methyladenosine–binding proteins suppress HIV-1 infectivity and viral production.
      ) (Fig. 4A). These studies provide a new therapeutic thought for treatment of AIDS.
      The m6A modifications on viral RNAs installed by host m6A writers can also serve as a molecular marker for host to distinguish self and non-self RNA in innate immune response (
      • Lu M.J.
      • Zhang Z.J.
      • Xue M.G.
      • Zhao B.X.S.
      • Harder O.
      • Li A.Z.
      • Liang X.Y.
      • Gao T.Z.
      • Xu Y.S.
      • Zhou J.Y.
      • Feng Z.D.
      • Niewiesk S.
      • Peeples M.E.
      • He C.
      • Li J.R.
      N6-methyladenosine modification enables viral RNA to escape recognition by RNA sensor RIG-I.
      ). The host utilizes several pattern-recognition receptors to sense foreign RNAs, such as retinoic-acid-inducible gene I-like receptors (RIG-I) and melanoma differentiation-associated gene 5 (MDA5). Both of them can sense non-self-viral RNA species and activate mitochondrial antiviral signaling, leading to the expression of type I and III interferons (IFNs) and antiviral response (
      • Chan Y.K.
      • Gack M.U.
      Viral evasion of intracellular DNA and RNA sensing.
      ). RNA modification 2′-O-methylation in viral RNA can escape the RNA sensor MDA5 (
      • Ringeard M.
      • Marchand V.
      • Decroly E.
      • Motorin Y.
      • Bennasser Y.
      FTSJ3 is an RNA 2′-O-methyltransferase recruited by HIV to avoid innate immune sensing.
      ). Similarly, m6A can also be hijacked by virus to escape the host RIG-I sensing machinery. The results from human metapneumovirus (HMPV), Hepatitis B virus (HBV), and HCV indicate that m6A in these viral RNA decreases the binding of RIG-I and promotes virus replication while m6A deficiency in these viral RNA increases RIG-I binding and the expression of type I IFNs (
      • Kim G.-W.
      • Imam H.
      • Khan M.
      • Siddiqui A.
      N6-methyladenosine modification of hepatitis B and C viral RNAs attenuates host innate immunity via RIG-I signaling.
      ).
      Studies of m6A writers and erasers indicate that m6A can also directly regulate gene expression of host genes involved in antiviral immune signaling. Upon vesicular stomatitis virus (VSV) infection, DEAD-box helicase 46 (DDX46) recruits m6A demethylase ALKBH5 onto its binding transcripts encoding antiviral proteins MAVS, TRAF3, and TRAF6 for m6A demethylation, which leads to the nuclear retention of these transcripts and inhibits the production of IFN-I (
      • Zheng Q.L.
      • Hou J.
      • Zhou Y.
      • Li Z.Y.
      • Cao X.T.
      The RNA helicase DDX46 inhibits innate immunity by entrapping m6A-demethylated antiviral transcripts in the nucleus.
      ) (Fig. 4A). Besides, Alkbh5-deficient mice display viral resistance through the regulation of metabolite itaconate, an important immune-activated metabolic enzyme, which is required for the effective infection of macrophages and epithelial cells by virus via an IFN-I independent mechanism (
      • O’Neill L.A.
      • Artyomov M.N.
      Itaconate: The poster child of metabolic reprogramming in macrophage function.
      ,
      • Domínguez-Andrés J.
      • Novakovic B.
      • Li Y.
      • Scicluna B.P.
      • Gresnigt M.S.
      • Arts R.J.
      • Oosting M.
      • Moorlag S.J.
      • Groh L.A.
      • Zwaag J.
      The itaconate pathway is a central regulatory node linking innate immune tolerance and trained immunity.
      ). α-KG dehydrogenase (OGDH) participates in tricarboxylic acid (TCA) cycle, which influences the production of itaconate, and m6A modification in OGDH mRNA is the target of ALKBH5. The depletion of ALKBH5 increases more m6A in OGDH mRNA and decreases OGDH expression through m6A-mediated mRNA degradation, thereby reducing the production of itaconate and inhibiting viral replication (
      • Liu Y.
      • You Y.L.
      • Lu Z.K.
      • Yang J.
      • Li P.P.
      • Liu L.
      • Xu H.N.
      • Niu Y.M.
      • Cao X.T.
      N6-methyladenosine RNA modification–mediated cellular metabolism rewiring inhibits viral replication.
      ). Additionally, heterogeneous nuclear ribonucleoproteins A2/B1 (hnRNPA2B1) as an m6A-binding protein plays regulatory roles in initiation and enhancement of the innate immune response to DNA virus herpes simplex virus-1 (HSV-1) and interacts with FTO. Upon HSV-1 infection, hnRNPA2B1 breaks the interaction with FTO to retain more m6A in CGAS, IFl16, and STING mRNA and promotes nuclear exports and the translation of these transcripts, therefore enhancing STING-dependent cytoplasmic antiviral signaling (
      • Wang L.
      • Wen M.Y.
      • Cao X.T.
      Nuclear hnRNPA2B1 initiates and amplifies the innate immune response to DNA viruses.
      ). These findings demonstrate that m6A on the host transcripts related to antiviral immune pathway can affect the stability of the transcripts, thereby regulating gene expression and antiviral ability of the host.
      The Coronavirus Disease 2019 (COVID-19) has become a pandemic in the world since December 2019, which is caused by Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) (
      • Gorbalenya A.
      • Baker S.
      • Baric R.
      • de Groot R.
      • Drosten C.
      • Gulyaeva A.
      • Haagmans B.
      • Lauber C.
      • Leontovich A.
      • Neuman B.
      The species severe acute respiratory syndrome related coronavirus: Classifying 2019-nCoV and naming it SARS-CoV-2.
      ). SARS-CoV-2 has a 30 kb-length single-stranded, positive-sense genomic RNA (
      • Zhou P.
      • Yang X.L.
      • Wang X.G.
      • Hu B.
      • Zhang L.
      • Zhang W.
      • Si H.R.
      • Zhu Y.
      • Li B.
      • Huang C.L.
      A pneumonia outbreak associated with a new coronavirus of probable bat origin.
      ). After the entry of SARS-CoV-2, the full-length and subgenomic negative-sense RNAs are synthesized from the positive-sense gRNA and serve as templates for progeny viral RNA synthesis and mRNA synthesis, respectively. Recent findings suggest that the m6A modification plays regulatory role in SARS-CoV-2 infection. The host m6A writer complex containing METTL3/METTL14 key subunits installs m6A in SARS-CoV-2 gRNA and the negative-sense RNA (
      • Liu J.E.
      • Xu Y.P.
      • Li K.
      • Ye Q.
      • Zhou H.Y.
      • Sun H.
      • Li X.
      • Yu L.
      • Deng Y.Q.
      • Li R.T.
      • Cheng M.L.
      The m6A methylome of SARS-CoV-2 in host cells.
      ). SARS-CoV-2 infection leads to the nuclear subcellular localization of the host m6A writer METTL14 and eraser ALKBH5 into the cytoplasm in Huh7 cells. The replication of SARS-CoV-2 increases upon knockdown of m6A writer METTL3, METTL14, and m6A reader YTH domain factor 2 (YTHDF2), but decreases after knockdown of m6A eraser ALKBH5, showing that m6A inhibits SARS-CoV-2 replication (
      • Liu J.E.
      • Xu Y.P.
      • Li K.
      • Ye Q.
      • Zhou H.Y.
      • Sun H.
      • Li X.
      • Yu L.
      • Deng Y.Q.
      • Li R.T.
      • Cheng M.L.
      The m6A methylome of SARS-CoV-2 in host cells.
      ).
      The m6A modification also regulates host cell innate immune responses during SARS-CoV-2 infection. RIG-I interacts with SARS-CoV-2 gRNA, but not m6A-modified SARS-CoV-2 gRNA. Knockdown of METTL3 reduces m6A methylation in SARS-CoV-2 gRNA and increases RIG-I binding to SARS-CoV-2 gRNA, thereby triggering the downstream innate immune signaling pathway and inflammatory gene expression (Fig. 4A). Consistently, the patients with severe COVID-19 exhibit the decreased METTL3 expression levels and induced inflammatory genes expression (
      • Li N.
      • Hui H.
      • Bray B.
      • Gonzalez G.M.
      • Zeller M.
      • Anderson K.G.
      • Knight R.
      • Smith D.
      • Wang Y.
      • Carlin A.F.
      METTL3 regulates viral m6A RNA modification and host cell innate immune responses during SARS-CoV-2 infection.
      ). Collectively, these results suggest that m6A plays dual functions in SARS-CoV-2 infection: the host uses m6A writer to methylate SARS-CoV-2 gRNA for degradation through YTHDF2-mediated mRNA decay; however, m6A is also hijacked by SARS-CoV-2 to escape the host RIG-I sensing machinery.

      Cancer progression

      In different types of cancer, the aberrant expression of m6A writers or erasers results in a significant change of m6A levels in cancer cells and thus influences oncogenesis, including cell adhesion, proliferation, invasion, and apoptosis (
      • Li E.
      • Wei B.
      • Wang X.
      • Kang R.
      METTL3 enhances cell adhesion through stabilizing integrin β1 mRNA via an m6A-HuR-dependent mechanism in prostatic carcinoma.
      ,
      • Xia T.
      • Wu X.
      • Cao M.
      • Zhang P.
      • Shi G.
      • Zhang J.
      • Lu Z.
      • Wu P.
      • Cai B.
      • Miao Y.
      The RNA m6A methyltransferase METTL3 promotes pancreatic cancer cell proliferation and invasion.
      ,
      • Li F.
      • Zhang C.
      • Zhang G.
      m6A RNA methylation controls proliferation of human glioma cells by influencing cell apoptosis.
      ).
      In glioblastoma (GBM), stem cells are considered as a new promising therapy target (
      • Chai R.C.
      • Wu F.
      • Wang Q.X.
      • Zhang S.
      • Zhang K.N.
      • Liu Y.Q.
      • Zhao Z.
      • Jiang T.
      • Wang Y.Z.
      • Kang C.S.
      m6A RNA methylation regulators contribute to malignant progression and have clinical prognostic impact in gliomas.
      ). ALKBH5 is highly expressed in glioblastoma stem cells (GSCs) and is required for their proliferation and tumorigenesis, and knockdown of ALKBH5 inhibits the proliferation of patient-derived GSCs. ALKBH5 demethylates m6A-modified FOXM1 pre-mRNA, a key cell-cycle molecule that is required for the transition between G1/S and G2/M during cell division (
      • Li Y.
      • Zhang S.
      • Huang S.
      FoxM1: A potential drug target for glioma.
      ). The m6A demethylation on FOXM1 by ALKBH5 affects the interaction between FOXM1 pre-mRNA and HuR, a nuclear RNA binding protein, and subsequently stabilizes FOXM1 expression levels (
      • Zhang S.C.
      • Zhao B.X.S.
      • Zhou A.D.
      • Lin K.Y.
      • Zheng S.P.
      • Lu Z.K.
      • Chen Y.H.
      • Sulman E.P.
      • Xie K.P.
      • Bögler O.
      m6A demethylase ALKBH5 maintains tumorigenicity of glioblastoma stem-like cells by sustaining FOXM1 expression and cell proliferation program.
      ) (Fig. 4B). Moreover, METTL3 is also elevated in GSCs and attenuated during differentiation. Specifically, METTL3 increases the m6A methylation of the transcription factor SOX2 mRNA, which controls a number of genes involved in embryonic development, and enhances its stability by recruiting HuR and promotes the maintenance and radio-resistance of GSCs (
      • Visvanathan A.
      • Patil V.
      • Arora A.
      • Hegde A.
      • Arivazhagan A.
      • Santosh V.
      • Somasundaram K.
      Essential role of METTL3-mediated m6A modification in glioma stem-like cells maintenance and radioresistance.
      ) (Fig. 4B). However, in these two studies, HuR plays opposite binding property, preferentially binds m6A-unmodified and -modified RNA substrates, respectively, which depends on the distance between the HuR-binding site and m6A site. In addition, METTL3 alters A-to-I and C-to-U RNA editing events by posttranscriptionally regulating the RNA editing enzymes ADAR and APOBEC3A and manipulates m6A modification on lncRNAs in GSCs. The silencing of METTL3 boosts several aberrant alternative splicing events, which indicates that METTL3 is able to determine several steps in RNA processing and to fine-tune the expressions of genes involved in the oncogenic pathway in GSCs (
      • Visvanathan A.
      • Patil V.
      • Abdulla S.
      • Hoheisel J.D.
      • Somasundaram K.
      N6-methyladenosine landscape of glioma stem-like cells: METTL3 is essential for the expression of actively transcribed genes and sustenance of the oncogenic signaling.
      ).
      In some subtypes of acute myeloid leukemia (AML), m6A writers and erasers are typically upregulated and the inactivation of the m6A machinery proteins results in restricted cell proliferation. FTO is highly expressed in AMLs with t(11q23)/MLL rearrangements, t(15;17)/PML-RARA, FLT3-ITD, and/or NPM1 mutations (
      • Li Z.J.
      • Weng H.Y.
      • Su R.
      • Weng X.C.
      • Zuo Z.X.
      • Li C.Y.
      • Huang H.L.
      • Nachtergaele S.
      • Dong L.
      • Hu C.
      FTO plays an oncogenic role in acute myeloid leukemia as a N6-methyladenosine RNA demethylase.
      ). The highly expressed FTO reduces m6A in mRNA transcripts ankyrin repeat and SOCS box containing 2 (ASB2) and retinoic acid receptor alpha (RARA) and subsequently decreases the transcript expression levels of ASB2 and RARA, thereby enhancing leukemic oncogene-mediated cell transformation and leukemogenesis and inhibiting all-trans-retinoic acid (ATRA)-induced AML cell differentiation (
      • Li Z.J.
      • Weng H.Y.
      • Su R.
      • Weng X.C.
      • Zuo Z.X.
      • Li C.Y.
      • Huang H.L.
      • Nachtergaele S.
      • Dong L.
      • Hu C.
      FTO plays an oncogenic role in acute myeloid leukemia as a N6-methyladenosine RNA demethylase.
      ) (Fig. 4B). However, m6A readers YTHDF1 and YTHDF2 have no obvious effect on the regulation of ASB2 and RARA, suggesting that the reader that promotes the mRNA stability of FTO target transcripts in AML has yet to be identified. The oncogenic role of the demethylation of FTO in AML is further confirmed by small-molecule inhibitor FB23-2 selectively targeting FTO demethylation activity (
      • Huang Y.
      • Su R.
      • Sheng Y.
      • Dong L.
      • Dong Z.
      • Xu H.
      • Ni T.
      • Zhang Z.S.
      • Zhang T.
      • Li C.
      Small-molecule targeting of oncogenic FTO demethylase in acute myeloid leukemia.
      ). METTL3 and METTL14 display their oncogenic roles by promoting the expression of oncogenes such as MYB and MYC through m6A modification (
      • Weng H.Y.
      • Huang H.L.
      • Wu H.Z.
      • Qin X.
      • Zhao B.X.S.
      • Dong L.
      • Shi H.L.
      • Skibbe J.
      • Shen C.
      • Hu C.
      • Sheng Y.
      • Wang Y.G.
      • Wunderlich M.
      • Zhang B.
      • Dore L.C.
      • et al.
      METTL14 inhibits hematopoietic stem/progenitor differentiation and promotes leukemogenesis via mRNA m6A modification.
      ,
      • Vu L.P.
      • Pickering B.F.
      • Cheng Y.
      • Zaccara S.
      • Nguyen D.
      • Minuesa G.
      • Chou T.
      • Chow A.
      • Saletore Y.
      • MacKay M.
      • Schulman J.
      • Famulare C.
      • Patel M.
      • Klimek M.V.
      • Garrett-Bakelman F.E.
      • et al.
      The N6-methyladenosine (m6A)-forming enzyme METTL3 controls myeloid differentiation of normal hematopoietic and leukemia cells.
      ). Additionally, METTL3 binds to promoters for m6A methylation and functions as an essential gene for leukemia growth (
      • Barbieri I.
      • Tzelepis K.
      • Pandolfini L.
      • Shi J.
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      Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.
      ). Mechanism study reveals that METTL3 alone is recruited to bind promoters of specific genes (e.g., SP1) by CAATT-box binding protein CEBPZ and subsequently installs m6A modifications within the coding region of these specific genes, thereby enhancing translation of these mRNAs by relieving ribosome stalling at GAN codons (N = A, U, C or G) (
      • Barbieri I.
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      Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.
      ) (Fig. 4B). Although CEBPZ can recruit METTL3 and guide it to specific transcripts for m6A deposition, it does not belong to m6A methyltransferase complex.
      In addition, ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in AML by influencing the stability of target mRNA in YTHDF2-dependent manner, including TACC3, AXL, and KDM4C transcripts (
      • Wang J.Z.
      • Li Y.C.
      • Wang P.P.
      • Han G.Q.
      • Zhang T.T.
      • Chang J.W.
      • Yin R.
      • Shan Y.
      • Wen J.
      • Xie X.Q.
      • Feng M.D.
      • Wang Q.F.
      • Hu J.
      • Cheng Y.
      • Zhang T.
      • et al.
      Leukemogenic chromatin alterations promote AML leukemia stem cells via a KDM4C-ALKBH5-AXL signaling axis.
      ,
      • Shen C.
      • Sheng Y.
      • Zhu A.C.
      • Robinson S.
      • Jiang X.
      • Dong L.
      • Chen H.Y.
      • Su R.
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      • Weng H.Y.
      • Huang H.L.
      • et al.
      RNA demethylase ALKBH5 selectively promotes tumorigenesis and cancer stem cell self-renewal in acute myeloid leukemia.
      ). KDM4C, an H3K9me3 demethylase, can regulate ALKBH5 expression by increasing the chromatin accessibility toward ALKBH5, which triggers the subsequent interaction between ALKBH5 and the target mRNA (Fig. 4B). METTL3, METTL14, FTO, and ALKBH5 are all upregulated in AML and promote cell growth, which seems a little contradictory. However, it is reasonable considering that the distinguishing downstream targets of these m6A machinery proteins in different cancer context or different subtypes of AML determine the different directions of cell fate.
      m6A writers or erasers are highly expressed in some cancers and promote cell proliferation and tumorigenesis. Nevertheless, in certain cancer cells, either m6A writers or erasers are downregulated and play an important role during oncogenesis (
      • Wang T.Y.
      • Kong S.
      • Tao M.
      • Ju S.Q.
      The potential role of RNA N6-methyladenosine in cancer progression.
      ). For example, in endometrial cancer, METTL3 and METTL14 are either downregulated or mutated, which consequently reduces m6A abundance. The decreased expression of the negative AKT regulator PHLPP2 and increased expression of the positive AKT regulator mTORC2 mediating by YTHDF2 and YTHDF1 respectively lead to the activation of the AKT pathway, which results in increased proliferation and tumorigenicity (
      • Liu J.
      • Eckert M.A.
      • Harada B.T.
      • Liu S.-M.
      • Lu Z.
      • Yu K.
      • Tienda S.M.
      • Chryplewicz A.
      • Zhu A.C.
      • Yang Y.
      m6A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.
      ) (Fig. 4B). Furthermore, in pancreatic cancer, ALKBH5 is downregulated and inhibits cell proliferation or motility by mediating Wnt signaling through the demethylation of Wnt inhibitory factor 1 (WIF-1) and decreasing the methylation and expression of the lncRNA KCNK15-AS1 (
      • He Y.
      • Hu H.
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      • Yuan H.
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      • Wu P.F.
      • Liu D.F.
      • Tian L.
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      • Jiang K.R.
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      ALKBH5 inhibits pancreatic cancer motility by decreasing long non-coding RNA KCNK15-AS1 methylation.
      ,
      • Tang B.
      • Yang Y.
      • Kang M.
      • Wang Y.
      • Wang Y.
      • Bi Y.
      • He S.
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      m6A demethylase ALKBH5 inhibits pancreatic cancer tumorigenesis by decreasing WIF-1 RNA methylation and mediating Wnt signaling.
      ). In general, m6A modifications on different tumor suppressor genes or oncogenes are affected by either m6A writers or erasers and recognized by diversified m6A readers for gene regulation.
      Based on the studies of m6A writers and erasers in cancer progression and viral infection, m6A displays complexity in regulation the occurrence of diseases. The basic functional mechanism of m6A modification in diseases is that the significant change of specific m6A on disease-related transcripts affects gene regulation through m6A readers-mediated RNA processing and metabolism, thereby executing corresponding downstream functions. However, the current studies on the biological functions of m6A are still not completely elucidated. Although the research on viral infection and cancer progression has reached single-transcript and single-base level, some reverse verification methods are still needed to prove the authenticity of the results.

      Programmable m6A editing based on m6A writers/erasers-conjugated CRISPR/Cas system

      Current evidence suggests that the specific m6A content is extremely important for maintaining normal physiological processes in mammals. In cancer, the higher or lower expression of m6A methyltransferase and demethylase can either promote or suppress tumorigenesis by affecting the expression of specific oncogenes or tumor suppressor genes. Therefore, the regulation of m6A modification at specific sites is of great significance for deeply studying the biological functions associated with m6A and to promote effective therapeutic strategies for cancer and other diseases. In recent 2 years, the combination of the CRISPR/Cas system and m6A writers or erasers has successfully achieved site-specific m6A editing in single transcript (
      • Liu X.-M.
      • Zhou J.
      • Mao Y.
      • Ji Q.
      • Qian S.-B.
      Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates.
      ,
      • Ying X.L.
      • Jiang X.
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      • Huang Y.P.
      • Zhu X.W.
      • Qi D.F.
      • Yuan G.
      • Luo J.H.
      • Ji W.D.
      Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development.
      ,
      • Wilson C.
      • Chen P.J.
      • Miao Z.
      • Liu D.R.
      Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase.
      ,
      • Li J.X.
      • Chen Z.J.
      • Chen F.
      • Xie G.Y.
      • Ling Y.Y.
      • Peng Y.x.
      • Lin Y.
      • Luo N.
      • Chiang C.M.
      • Wang H.S.
      Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.
      ,
      • Liu J.
      • Dou X.Y.
      • Chen C.Y.
      • Chen C.
      • Liu C.
      • Xu M.M.
      • Zhao S.Q.
      • Shen B.
      • Gao Y.W.
      • Han D.L.
      • He C.
      N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.
      ,
      • Mo J.
      • Chen Z.G.
      • Qin S.S.
      • Li S.
      • Liu C.G.
      • Zhang L.
      • Ran R.X.
      • Kong Y.
      • Wang F.
      • Liu S.M.
      • Zhou Y.
      • Zhang X.L.
      • Weng X.C.
      • Zhou X.
      TRADES: Targeted RNA demethylation by SunTag system.
      ,
      • Zhao J.
      • Li B.
      • Ma J.X.
      • Jin W.L.
      • Ma X.L.
      Photoactivatable RNA N6-methyladenosine editing with CRISPR-Cas13.
      ).
      The CRISPR/Cas system is a natural immune mechanism that allows for bacteria to resist viral infection (
      • Mojica F.J.
      • Díez-Villaseñor C.
      • García-Martínez J.
      • Soria E.
      Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.
      ). It is now regarded the most powerful gene editing tool that is able to achieve simple knockout of almost all genes in eukaryotes (
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • Hsu P.D.
      • Wu X.
      • Jiang W.
      • Marraffini L.A.
      • Zhang F.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Mali P.
      • Yang L.
      • Esvelt K.M.
      • Aach J.
      • Guell M.
      • DiCarlo J.E.
      • Norville J.E.
      • Church G.M.
      RNA-guided human genome engineering via Cas9.
      ). Due to the excellent ability to target genomic DNA, the catalytically inactive CRISPR/Cas as a guide system is combined with other proteins and applied in single-base editing, chromatin engineering, cell imaging, and epigenetic editing (
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.Y.
      • Zhang D.L.
      • Liu Z.
      • Gong Z.
      • Wang Q.
      • Huang J.B.
      • Tang C.
      • Zou T.T.
      • Yin P.
      Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex.
      ,
      • Komor A.C.
      • Kim Y.B.
      • Packer M.S.
      • Zuris J.A.
      • Liu D.R.
      Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.
      ,
      • Deng W.
      • Rupon J.W.
      • Krivega I.
      • Breda L.
      • Motta I.
      • Jahn K.S.
      • Reik A.
      • Gregory P.D.
      • Rivella S.
      • Dean A.
      • Blobel G.A.
      Reactivation of developmentally silenced globin genes by forced chromatin looping.
      ,
      • Chen B.
      • Gilbert L.A.
      • Cimini B.A.
      • Schnitzbauer J.
      • Zhang W.
      • Li G.-W.
      • Park J.
      • Blackburn E.H.
      • Weissman J.S.
      • Qi L.S.
      • Huang B.
      Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.
      ). For example, the CRISPR/Cas9 system is able to mediate epigenetic editing by fusing catalytically dead Cas9 (dCas9) to the corresponding epigenetic modification enzymes, such as DNMT3A for 5 mC methylation, TET1 for 5 mC demethylation, or LSD1 for histone modification H3K4me2 demethylation (
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.Y.
      • Zhang D.L.
      • Liu Z.
      • Gong Z.
      • Wang Q.
      • Huang J.B.
      • Tang C.
      • Zou T.T.
      • Yin P.
      Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex.
      ,
      • Kearns N.A.
      • Pham H.
      • Tabak B.
      • Genga R.M.
      • Silverstein N.J.
      • Garber M.
      • Maehr R.
      Functional annotation of native enhancers with a Cas9–histone demethylase fusion.
      ).
      To create diverse ability on targeting RNA, two kinds of CRISPR/Cas systems have been developed to allow cutting target RNAs. CRISPR/Cas9 system was modified to specifically recognize and cut RNA by adding a protospacer adjacent motif (PAM)-presenting antisense oligonucleotide (PAMmer), known as RNA-guided Cas9 (RCas9) (
      • O’Connell M.R.
      • Oakes B.L.
      • Sternberg S.H.
      • East-Seletsky A.
      • Kaplan M.
      • Doudna J.A.
      Programmable RNA recognition and cleavage by CRISPR/Cas9.
      ). Cas13, a type Ⅳ CRISPR/Cas system protein, assembles with crispr RNA (crRNA) and forms a crRNA-guided RNA targeting effector complex to directly cut RNA (
      • Cox D.B.T.
      • Gootenberg J.S.
      • Abudayyeh O.O.
      • Franklin B.
      • Kellner M.J.
      • Joung J.
      • Zhang F.
      RNA editing with CRISPR-Cas13.
      ). Similarly, catalytically dead RCas9 (dRCas9) and Cas13 (dCas13) have been used as RNA-targeting tools to image RNA and to identify interaction proteins of RNA (
      • Yang L.-Z.
      • Wang Y.
      • Li S.-Q.
      • Yao R.-W.
      • Luan P.-F.
      • Wu H.
      • Carmichael G.G.
      • Chen L.-L.
      Dynamic imaging of RNA in living cells by CRISPR-Cas13 systems.
      ,
      • Han S.
      • Zhao B.S.
      • Myers S.A.
      • Carr S.A.
      • He C.
      • Ting A.Y.
      RNA–protein interaction mapping via MS2-or Cas13-based APEX targeting.
      ). Recently, combining with m6A writers or erasers, these two systems are successfully applied into programming RNA modification (Fig. 4C).
      The fusion of dRCas9 with either MTase domain of METTL3 and METTL14 or with m6A demethylases ALKBH5 or FTO forms engineered m6A writers or erasers to install or delete m6A at specific sites in transcripts. Site-specific addition of m6A at 5′ UTR or 3′ UTR in mRNA regulates downstream translation or mRNA stability, and site-specific removal of m6A in lncRNA MALAT1 affects RNA structure to regulate the binding affinity of MALAT1-interaction protein HNRNPC (
      • Liu X.-M.
      • Zhou J.
      • Mao Y.
      • Ji Q.
      • Qian S.-B.
      Programmable RNA N6-methyladenosine editing by CRISPR-Cas9 conjugates.
      ). Furthermore, the single catalytical domain of METTL3 fused with dRCas9 also installs site-specific m6A efficiently and has been used to write m6A onto the 3′ UTR of CDCP1 transcript in order to promote mRNA translation and bladder cancer development (
      • Ying X.L.
      • Jiang X.
      • Zhang H.Q.
      • Liu B.X.
      • Huang Y.P.
      • Zhu X.W.
      • Qi D.F.
      • Yuan G.
      • Luo J.H.
      • Ji W.D.
      Programmable N6-methyladenosine modification of CDCP1 mRNA by RCas9-methyltransferase like 3 conjugates promotes bladder cancer development.
      ).
      RCas9 system needs additional artificially synthesized PAMmer to target RNA, which makes the RCas9 system more complicated and incompatible with some delivery strategies, such as viral infection. The Cas13 protein, which directly targets RNA with a guide RNA, is able to avoid these drawbacks and provides a new tool for RNA editing. In particular, dCas13 fused to the METTL3 protein lacking its zinc finger domain is able to target single RNA and install m6A modification. The dCas13-METTL3 m6A editing system can be fused with either a nuclear localization signal sequence or a nuclear export signal sequence to edit specific m6A sites in nuclear RNA or cytoplasmic mature transcripts (
      • Wilson C.
      • Chen P.J.
      • Miao Z.
      • Liu D.R.
      Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase.
      ). The engineered m6A erasers are also developed through the combination of dCas13 with m6A demethylases. The fusion of dCas13 and ALKBH5 (dm6ACRISPR) is used to demethylate specific m6A sites on EGFR and MYC transcripts to inhibit the proliferation of cancer cells (
      • Li J.X.
      • Chen Z.J.
      • Chen F.
      • Xie G.Y.
      • Ling Y.Y.
      • Peng Y.x.
      • Lin Y.
      • Luo N.
      • Chiang C.M.
      • Wang H.S.
      Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.
      ), and the fusion of dCas13 and FTO can also remove m6A in nuclear RNA LINE-1 in order to affect transcription (
      • Liu J.
      • Dou X.Y.
      • Chen C.Y.
      • Chen C.
      • Liu C.
      • Xu M.M.
      • Zhao S.Q.
      • Shen B.
      • Gao Y.W.
      • Han D.L.
      • He C.
      N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.
      ).
      SunTag system can realize the recruitment of multiple protein copies to a polypeptide scaffold (
      • Tanenbaum M.E.
      • Gilbert L.A.
      • Qi L.S.
      • Weissman J.S.
      • Vale R.D.
      A protein-tagging system for signal amplification in gene expression and fluorescence imaging.
      ). dCas13 coupled with the SunTag system acts as another type of m6A eraser tool, termed as TRADES. Specifically, dCas13 fused with ten copies of GCN4 peptides can recruit multiple scFv-FTO or ALKBH5 fusion proteins to demethylate the target m6A, which increases the demethylation window (
      • Mo J.
      • Chen Z.G.
      • Qin S.S.
      • Li S.
      • Liu C.G.
      • Zhang L.
      • Ran R.X.
      • Kong Y.
      • Wang F.
      • Liu S.M.
      • Zhou Y.
      • Zhang X.L.
      • Weng X.C.
      • Zhou X.
      TRADES: Targeted RNA demethylation by SunTag system.
      ). Furthermore, photoactivatable m6A editing tools have also been developed using the blue-light-inducible heterodimer proteins CIBN and CRY2PHR. In this system, CIBN and CRY2PHR are respectively fused to dCas13 and m6A writer or erasers. Under blue light, CIBN and CRY2PHR form a heterodimer to recruit m6A writer or erasers at the dCas13-guided regions for m6A editing (
      • Zhao J.
      • Li B.
      • Ma J.X.
      • Jin W.L.
      • Ma X.L.
      Photoactivatable RNA N6-methyladenosine editing with CRISPR-Cas13.
      ).
      Site-specific regulation of m6A has achieved precise m6A editing in cells. Compared with rough overexpression of m6A writers or erasers, the utility of CRISPR/Cas system provides better targeting ability for the addition or removal of m6A on specific sites and avoids the interference of global m6A, which make the results more reliable when studying the biological functions of m6A. However, in order to achieve the precise control of m6A editing and solve the off-target effect rising from CRISPR/Cas system itself, more accurate tools are needed for comprehensively and deeply studying the physiological functions of m6A and treating diseases.

      Conclusions and perspectives

      The m6A modification in RNA participates in various biological processes. The normal performance of these biological processes is closely related to the levels of m6A and their correct regulation. Therefore, studying the distribution patterns of m6A in cells is of great significance for the exploration of the biological functions associated with m6A. The emergence of m6A-specific antibody provides an effective method for studying the transcriptome-wide distribution of m6A. However, due to a high false-positive rate, poor reproducibility and low resolution, it is imperative to develop more accurate m6A detection techniques. The in-depth exploration of the mechanisms of m6A methyltransferase and demethylase has allowed to develop new m6A sequencing methods assisted by m6A writers and erasers, which greatly improved the authenticity and sensitivity of m6A detection. However, all available methods have not fully met the needs for quantification of m6A fraction, single-base resolution, and low amount of RNA (from nanograms of RNA to single cell) at the same time.
      In recent years, the mechanistic role of m6A in different diseases (e.g., cancer) has been gradually explained. Many cancers are caused by the abnormal expression of m6A writers or erasers in cells, which varies the m6A fractions of some specific transcripts and subsequently affects gene expression, leading to the occurrence and development of diseases. Therefore, adjusting the level of m6A on specific transcripts might facilitate the maintenance of homeostasis in transcripts and impact biological functions. The development of engineered m6A editing tools assisted by RNA guider dRCas9 or dCas13 provides an alternative method for site-specific m6A regulation, which can be applied for m6A functional study and potential tools for treatment of diseases.
      Unlike the direct regulation of RNA expression through knockdown or overexpression, the regulation of m6A in specific transcripts can allow for the posttranscriptional regulation of RNA. The function of m6A is far from simple and is determined by the m6A-binding proteins nearby (
      • Zhang Z.
      • Luo K.
      • Zou Z.
      • Qiu M.
      • Tian J.
      • Sieh L.
      • Shi H.
      • Zou Y.
      • Wang G.
      • Morrison J.
      Genetic analyses support the contribution of mRNA N6-methyladenosine (m6A) modification to human disease heritability.
      ). m6A can be regarded as a multifunctional button that determines the fate of RNA molecules. By regulating the levels of m6A in the transcript, organisms can make corresponding stress responses under different physiological conditions and achieve the autonomous regulation of transcript structure and expression to be able to adapt to changes in the environment.

      Conflict of interest

      The authors have declared no conflicts of interest for this article.

      Acknowledgments

      This work was supported by Beijing Natural Science Foundation ( Z200010 ), the National Natural Science Foundation of China (nos. 21822702 , 92053109 , and 21820102008 ), and the National Basic Research Program of China ( 2019YFA0802201 and 2017YFA0505201 ).

      Author contributions

      W. Z and Y. Q. writing–original draft; G. J. supervision; G. J. funding acquisition; G. J. writing–review and editing.

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