BCM_Seminario_III-3_Gene Expression Regulation Mediated Through Reversible m6A RNA Methylation Nrg3724

In the central dogma of molecular biology, genetic infor-mation flows from DNA to RNA and then to protein. Reversible epigenetic modifications occur on genomic DNA15 and histone proteins69 to substantially regulate gene expression that defines cell status and that affects cell differentiation and development (FIG.1). Although both DNA and proteins are subject to reversible chemi-cal tuning, as we pointed out in 2010, a similar process on mRNA or other forms of RNA as the third compo-nent of the central dogma had been missing10. RNA has crucial roles in biological systems not only by passing genetic information from DNA to protein but also by regulating various biological processes. The diverse functions of RNA are accompanied by more than 100 chemical modifications1114, although the functions of most of these RNA modifications have remained a mys-tery. Most RNA species were thought to be short lived, and RNA modifications were considered to be static and unalterable after their covalent attachment. The cen-tral role of RNA in gene expression and the intrinsic chemical reversibility of certain types of RNA meth-ylation prompted us to raise the question of reversible RNA modifications in gene expression regulation10. In this Review, we discuss how, only in the past 23years, N6-methyladenosine (m6A) has been discovered as the first example of reversible RNA methylation15,16. We describe the transcriptome-wide distribution of m6A in mammalian systems17,18, the identification of pro-tein writers, erasers and readers for this dynamic RNA

methylation, and emerging functions for m6A in several mechanisms of post-transcriptional regulation of gene expression (FIG.1).

m6A RNA methylation in eukaryotesDiscovery and quantification of m6A in mRNAs and long non-coding RNAs in eukaryotes. Discovered in the 1970s, m6A is the most prevalent internal modification in polyadenylated mRNAs and long non-coding RNAs (lncRNAs) in higher eukaryotes19. m6A is widely con-served among eukaryotic species that range from yeast, plants, flies to mammals, as well as among viral RNAs with a nuclear phase2024. The identified sequence content of m6A obtained from mutational studies and substrate preference of the methyltransferase enzyme invitro2527 is [G/A/U][G>A]m6AC[U>A>C]. The amount of m6A in isolated RNA was estimated to be 0.10.4% of that of ade-nines (that is, ~35 m6A sites per mRNA) in mammals19,28 and ~0.25% in meiotic Saccharomyces cerevisiae29. The total amount of m6A in RNA can be probed by several methods, including two-dimensional thin layer chromatography30, dot-blot15 and high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLCQqQ-MS/MS)15,16. The femtomole sensitivity achieved by HPLCQqQ-MS/MS makes it a quantitative tool for monitoring m6A dynam-ics; the purity of mRNA is extremely important for this measurement because ribosomal RNA (rRNA), small nuclear RNA (snRNA) and tRNA also containm6A.

1Department of Chemistry and Institute for Biophysical Dynamics, The University of Chicago, 929 East 57th Street, Chicago, Illinois 60637, USA.2Cancer Research Center, Chaim Sheba Medical Center, Tel Hashomer 52621, Israel.3Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.Correspondence to C.H.email: [email protected]:10.1038/nrg3724Published online 25 March 2014

Epigenetic modificationsReversible chemical modifications on DNA and histones that regulate gene expression independently of the genome sequences and that are heritable through cell division.

Writers, erasers and readersEnzymes or proteins that add, remove or preferentially bind to the chemical modifications at designated DNA or RNA nucleotides and amino acid residues of histones.

Gene expression regulation mediated through reversible m6A RNA methylationYe Fu1, Dan Dominissini1,2,3, Gideon Rechavi2,3 and Chuan He1

Abstract | Cellular RNAs carry diverse chemical modifications that used to be regarded as static and having minor roles in fine-tuning structural and functional properties of RNAs. In this Review, we focus on reversible methylation through the most prevalent mammalian mRNA internal modification, N6-methyladenosine (m6A). Recent studies have discovered protein writers, erasers and readers of this RNA chemical mark, as well as its dynamic deposition on mRNA and other types of nuclear RNA. These findings strongly indicate dynamic regulatory roles that are analogous to the well-known reversible epigenetic modifications of DNA and histone proteins. This reversible RNA methylation adds a new dimension to the developing picture of post-transcriptional regulation of gene expression.

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NATURE REVIEWS | GENETICS VOLUME 15 | MAY 2014 | 293

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MethyltransferaseAn enzyme that transfers a methyl group to its substrate. Most methyltransferases use S-adenosyl-l-methionine (SAM) as the methyl donor.

Two-dimensional thin layer chromatographyA technique to separate and identify nucleosides on cellulose plates according to their differential migration patterns in two different solvents. The nucleoside is typically radiolabelled for detection.

High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry(HPLCQqQ-MS/MS). A liquid chromatography method coupled with triple-quadrupole tandem mass spectrometry, which can quantitatively and simultaneously monitor multiple molecular species according to their fragmentation patterns.

Distribution of m6A in mammalian mRNAs and long non-coding RNAs. Before 2012, the genome-wide dis-tribution of m6A was unknown, until two independent studies developed an m6A RNA immunoprecipitation approach followed by high-throughput sequencing (MeRIPseq) to map the m6A RNA methylomes with a ~100-nucleotide resolution17,18. Briefly, the isolated mRNA is fragmented, immunoprecipitated using an m6A-targeted antibody, ligated to sequencing adap-tors, reverse-transcribed to cDNA, amplified using PCR and subjected to high-throughput sequencing (FIG.2a). The resulting maps have shown that m6A is widely distributed in more than 7,000 mRNA and 300 non-coding RNA (ncRNA) transcripts in human cells, and is enriched around stop codons, in 3 untranslated regions (3UTRs) and within internal long exons (FIG.2b). Additionally, the presence of m6A in introns suggests that this modification can be added either before or at the same time as RNA splicing. Many m6A peaks are well conserved between humans and mice, and dynamic changes of certain peaks have been observed under dif-ferent stress conditions. A potential link between m6A and microRNA-target sites was also suggested18. Several lncRNAs contain m6A, which indicates that certain ncRNAs transcribed by RNA polymerase II are also sub-ject to m6A methylation. This approach has since been widely adopted in studies of transcriptome-wide m6A distribution.

Distribution of m6A in mRNA in meiotic yeast. In S.cerevisiae, m6A has an important role in the initiation of meiosis, which is induced by nitrogen starvation31. Although yeast cells lack m6A (or contain very little of it) in mRNA during the mitotic log phase, they begin to accumulate high levels of m6A during nitrogen starva-tion29. Genome-wide mapping of m6A has revealed 1,308 methylation sites in 1,183 transcripts in an ndt80-deficient (ndt80/) strain that was arrested during meiotic G2 phase or prophase32. The MeRIPseq approach was fur-ther optimized through the use of shorter mRNA frag-ments and an ime4/ strain (which lacks the inducer of meiosis4 (Ime4) methyltransferase) as a negative control to obtain a map of m6A at higher resolution. The methyl-ated transcripts were found to be less structured, and most of them encode functions that are particularly related to meiosis. These transcripts have a consensus sequence of ANRGm6ACNNU (where R denotes A or G, and N represents any nucleotide), and their methylation sites are enriched at the 3 ends, which is similar to those of the mammalian systems. Thus, the distribution pattern of m6A and the consensus sequence of these sites seem to be conserved to a large extent from yeast tohumans.

Quantitative detection of the m6A fraction with single-nucleotide resolution. The antibody-based profiling of m6A could not provide information at single-nucleotide resolution: although this method determines RNA frag-ments that harbour m6A, the difficulty in distinguishing m6A from unmodified adenines by sequencing hinders the pinpointing of m6A sites within these fragments. Multiple RRACH (where H denotes U, A or C) motifs could be present adjacent to each other, and the modi-fication may also occur at non-consensus sites. In addi-tion, antibody-based profiling cannot reveal the fraction of cellular RNAs that are modified at each specific site. Traditionally, radioactive labelling was used to detect the modification site; however, this procedure is long and tedious. A digestion-based method has recently been developed to determine the percentage of m6A at a specific site with single-nucleotide resolution33. Termed site-specific cleavage and radioactive labelling followed by ligation-assisted extraction and thin layer chromatog-raphy (SCARLET), this method uses RNase H guided by a sequence-specific 2-OMe/2-H chimeric oligonu-cleotide to cleave the 5 end of the candidate site for subsequent labelling and detection. Application of this method to two lncRNAs and three mRNAs revealed genuine m6A sites and quantified the methylation frac-tions (1177%). These results, together with the 20% of m6A modification that was previously reported in bovine prolactin mRNA34, indicate that many m6A sites in mRNA and lncRNA are incompletely methylated. In fact, the majority of m6A consensus sequence sites are not methylated in mammalian mRNA17,18.

Methylation on the N6 position of adenosine slightly reduces the stability of WatsonCrick A:U base pairing35, but it does not noticeably block the extension activities of most reverse transcriptases. Therefore, methods based on primer extension cannot be readily used to map precise modification positions.

Figure 1 | Reversible chemical modifications that regulate the flow of genetic information. In the central dogma, genetic information is passed from DNA to RNA and then to protein. Epigenetic DNA modifications (for example, the formation of 5-methylcytosine (m5C; also known as 5mC) and 5-hydroxymethylcytosine (hm5C; also known as 5hmC)) and histone modifications (for example, methylation (me) and acetylation (ac)) are known to have important roles in regulating cell differentiation and development. Reversible RNA modifications (for example, the formation of N6-methyladenosine (m6A) and N6-hydroxymethyladenosine (hm6A)) add an additional layer of dynamic regulation of biological processes.

Nature Reviews | Genetics

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However, Thermus thermophilus DNA polymerase I36 and HIV reverse transcriptase37 show kinetic differ-ences when extending opposite m6A compared with unmodified adenines, and they could be used to map m6A positions36,37. The application of this approach at

a genome scale is still limited, and a method for the genome-wide mapping of m6A with single-nucleotide resolution thus remains highly desirable.

m6A writers in eukaryotesMETTL3 is an active component of the m6A methyl-transferase complex in mammalian cells. m6A mRNA methylation is catalysed by a multicomponent methyl-transferase complex, which was originally isolated as ~200 kDa and ~800 kDa subcomplexes from HeLa nuclear extracts25,38. A 70 kDa protein METTL3 (known as MT-A70 when identified) was the only known com-ponent characterized38. METTL3 is highly conserved in eukaryotes from yeast to humans (FIG.3a). Knockdown of METTL3 in HeLa cells led to a ~30% decrease of the total m6A level, and the same knockdown experiment in HepG2 cells induced apoptosis, possibly through the activation of the p53-mediated pathway17,39. A recombi-nant FLAG-tagged human METTL3 protein has recently been shown to exhibit a low level of activity by itself. Other components are required to achieve optimal activity invitro40.

METTL14 is another active component of the m6A methyltransferase complex and forms a stable hetero complex with METTL3. A phylogenetic analysis of the METTL3 family of methyltransferases in the human genome identified METTL14 and METTL4 as close homologues of METTL3 with a conserved motif that contains either Asp-Pro-Pro-Trp or Glu-Pro-Pro-Leu41 (FIG.3a). We found that knockdown of METTL14, but not METTL4, leads to decreased m6A levels in HeLa and 293FT cells40. Biochemical characterization revealed that these two proteins form a stable complex with a stoichiometric ratio of 1:1. Although the methyl-ation activity of METTL14 is slightly higher than that of METTL3 invitro, the combination of both methyltrans-ferases leads to a substantially enhanced methylation activity. This heterodimer also shows a strong prefer-ence for the cognate m6A consensus sequence and a modest preference for less structured RNA invitro. METTL3 and METTL14 colocalize in nuclear speckles, and the heterodimer forms the core of the mamma-lian methyltransferase complex. Additional features of METTL14 include glycine-rich sequences in its car-boxyl terminus and a potential coiled-coil in its amino terminus (FIG.3a), which may participate in proteinprotein interactions in nuclear speckles. The binding sites of METTL3 and METTL14 in substrate RNAs, as shown by a photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PARCLIP) assay, contain a similar consensus sequence to that known for m6A (FIG.3b). Interestingly, silencing of the methyl-transferase complex led to an increase in the abundance and half-lives of their target RNAs, which is consistent with an emerging role for m6A as a negative regula-tor of gene expression (see below). A related study in mouse embryonic stem cells (mESCs) also indicates that METTL3 and METTL14 work as a complex42.

The discovery of the second active methyltransferase component in the core complex raises the following


cDNA library construction and high-throughput sequencing

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Figure 2 | Profiling of m6A in RNA by m6A RNA immunoprecipitation. Antibody-based N6-methyladenosine (m6A) RNA immunoprecipitation has been developed to profile the transcriptome-wide distribution of m6A. a | Isolated mRNA is fragmented to ~100 nucleotides, immunoprecipitated using m6A-specific antibodies, converted to a cDNA library and subjected to high-throughput sequencing. Comparison between the immunoprecipitated sample and the input sample identifies m6A signal peaks. b | Transcriptome-wide profiling of m6A in mRNA revealed that m6A is enriched around stop codons, at 3 untranslated regions and within long exons. The 5 cap contains the N6,2O-dimethyladenosine (m6Am) modification, which can also be enriched using the m6A-specific antibody. Me, methyl group.

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m6A RNA immunoprecipitationAn immunoprecipitation method to selectively enrich for N6-methyladenosine (m6A)-containing RNA using an m6A-targeted antibody.

Nuclear specklesNuclear domains located in the interchromatin regions of the nucleoplasm and enriched with pre-mRNA processing factors.

Photoactivatable ribonucle-oside-enhanced crosslinking and immunoprecipitation(PARCLIP). A biochemical method that takes advantage of incorporated photoreactive ribonucleoside analogues to identify the binding sites of RNA-binding proteins in cells.


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Fe(II)-bindingmotif (HXDXnH)

Substrate and -KGbinding (RXXXXXR)

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FTO

Figure 3 | Reversible m6A methylation of mRNA and other types of nuclear RNA. The N6-methyladenosine (m6A) modification is installed by a hetero complex of two methyltransferases METTL3METTL14, assisted by Wilms tumour1associating protein (WTAP), and can be demethylated by the -ketoglutarate (-KG)-dependent dioxygenases FTO and ALKBH5. a | Saccharomyces cerevisiae inducer of meiosis4 (Ime4), and human METTL3 and METTL14 contain the S-adenosyl-l-methionine (SAM)-dependent methyltransferase domain for m6A methylation. The (D/E)P(P/A)(W/L) active site and the SAMbinding motif are conserved. b | Photoactivatable ribonucleosideenhanced crosslinking and immunoprecipitation (PARCLIP) reveals that the binding sites of METTL14 and METTL3 on mRNA resemble the consensus sequence of m6A in mammalian mRNA. The sequence bound by WTAP moderately overlaps with those bound by METTL14 and METTL3. c | Mammalian FTO and ALKBH5 contain the active site motif HXDX

nH

(where X denotes any amino acid) for Fe(ii) binding, RXXXXXR for both -KG binding and substrate recognition, and an extra loop that leads to preferential binding of single-stranded over double-stranded nucleic acids68,121,122. Relative to Escherichia coli AlkB, mammalian ALKBH5 has an amino-terminal alanine-rich sequence and a potential coiled-coil structure that could be important for its localization. FTO contains an extra carboxy-terminal domain with a novel fold, possibly to engage in additional proteinprotein interactions. d | Methylation and demethylation of m6A on RNA are shown. Whereas ALKBH5 catalyses the direct removal of m6A, FTO can oxidize m6A to N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f6A) sequentially; hm6A and f6A are moderately stable (with halflives of ~3hours under physiological conditions) and can be hydrolysed to adenine.

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Yeast two-hybrid screensA method in which one protein is fused to the GAL4 activation domain and the other to the GAL4 DNA-binding domain, and both fusion proteins are introduced into yeast. Expression of a GAL4-regulated reporter gene indicates that the two proteins physically interact.

DemethylaseAn enzyme that removes a methyl group from its substrate.

question: why is the m6A methyltransferase complex composed of two active components, both of which bind to the methyl donor S-adenosyl-l-methionine (SAM)? The hetero complex may allow the selective tuning of methylation activity through post-translational modification of each component in order to affect dif-ferent substrate transcripts, thus having an influence on different biological pathways. A heterodimer of two methyltransferase components is required for optimal activities of several other known RNA methyltrans-ferase complexs4346. Typically, one subunit has a SAM-binding pocket, and the other non-catalytic subunit either stabilizes the catalytic subunit or enhances its activity by forming a continuous substrate-binding sur-face. However, both METTL3 and METTL14 are active. A crystal structure of this complex will be helpful in uncovering the synergy between the two enzymes and the properties associated with each active component.

WTAP is the third crucial component of the m6A methyl-transferase complex invivo. Yeast two-hybrid screens have identified FKBP12-interacting protein of 37 kDa (FIP37; also known as AT3g54170) in Arabidopsis thaliana47 and Mum2 in yeast as the partner proteins of the METTL3 homologues in these organisms48. These two proteins are homologues of the Wilms tumour 1-associating pro-tein (WTAP) in humans. WTAP was initially identified as a splicing factor that binds to the Wilms tumour1 (WT1) protein49, and it is essential for cell cycle progres-sion and early mammalian embryonic development. We found that knockdown of WTAP leads to a decrease in the total m6A level in HeLa and 293FT cells40. WTAP interacts with both METTL3 and METTL14, and colo-calizes with the METTL3METTL14 heterodimer in nuclear speckles to participate in m6A RNA methylation (FIG.3d). In fact, knockdown of WTAP leads to the largest decrease in m6A levels in these cell lines, which indicates that WTAP has important roles in cellular m6A depo-sition. A PARCLIP assay revealed that WTAP shares a similar binding sequence of GACU; that is, the sequence bound by WTAP moderately overlaps with the GGAC sequence bound by both METTL3 and METTL14 (FIG.3b). As identified by PARCLIP, these targets have a ~50% overlap with m6A-containing transcripts, which further indicates that METTL3, METTL14 and WTAP form the core of the major cellular writer complex of m6A (REF.40). A large proportion of the binding sites of these three proteins are found in introns (2934%), which fur-ther implies that methylation occurs co-transcriptionally either before or at the same time as splicing. As WTAP has been thought to be a splicing factor, a recent study indicates that the knockdown of WTAP or METTL3 yields different isoforms of m6A-containing transcripts, which suggests that methylation could affect splicing50.

How does WTAP enhance the methylation activity of METTL3 and METTL14 invivo (FIG.3d)? Potentially, WTAP may help to recruit METTL3 and METTL14 to their target mRNAs. WTAP has also been shown to be essential for the nuclear speckle localization of METTL3 and METTL14, which could affect the methylation efficiency of these proteins50. WTAP, which is known

to interact with many proteins and lncRNAs51, could also recruit other proteins or enzymes to the methyl-transferase complex; these additional factors may affect the methylation activity and selectivity through direct interactions or post-translational modifications. Future research to identify additional factors that interact with or modify the two methyltransferases is crucial for under-standing the selectivity of m6A deposition. We may then be able to answer questions such as how do cells choose to methylate certain RNA sites, and how is m6A tar-geted to 3UTRs and long exons. The potential interplay between RNA methylation, transcriptional regulation and splicing could also be further investigated.

Mum2Ime4Slz1 (MIS) complex in yeast mediates mRNA methylation during meiosis. Ime4 is the homo-logue of METTL3 in yeast and is crucial for the induction of yeast sporulation. Two other components of the meth-ylation complex Mum2 and sporulation specific with a leucine zipper motif protein 1 (Slz1) have been iden-tified through yeast two-hybrid experiments48. Mum2 is homologous to human WTAP, whereas Slz1 lacks mammalian homologues. Interestingly, the increase in m6A levels during meiosis is mainly triggered by Ime1 (a master regulator of meiosis), which transcriptionally induces SLZ1. Ime4 and Mum2 are expressed before the induction of meiosis, and Slz1 then recruits them from the cytoplasm to the nucleolus32. This nucleolar localiza-tion of the MIS complex is essential for accumulating the full level of m6A. In contrast to yeast, mammalian cells lack homologues of Slz1, and the mammalian and plant methyltransferase complex primarily locates in nuclear speckles instead of the nucleolus.

METTL3 and WTAP are highly conserved in eukary-otes. In A.thaliana, m6A seems to be mainly found near 3UTRs52; a mutation in MTA (which is the METTL3 homologue in A.thaliana) has been associated with cell division defects, arrested seeds, reduced apical dominance and organ abnormality47. In Drosophila melanogaster, the METTL3 homologue Ime4 is essen-tial for viability and regulates Notch signalling during egg chamber development53. In zebrafish, knockdown of either WTAP or METTL3 leads to multiple develop-mental defects, and knockdown of both proteins leads to increased apoptosis50.

m6A erasers in mammalsDemethylation of m6A in RNA by FTO. In 2011, the dis-covery of -ketoglutarate-dependent dioxygenase FTO as the first RNA demethylase was an important break-through in reigniting investigations of m6A biology15. The Fto gene was initially discovered in a deletion of four genes that led to a fused-toe phenotype in mutant mice54. In 2007, three independent studies revealed that a single-nucleotide polymorphism in the first intron of FTO strongly associates with body mass index and the risk of obesity in multiple populations5557. In adult mice, Fto has the highest expression level in the brain, particu-larly within the hypothalamus58. Deletion or overexpres-sion of Fto in mouse models has been associated with altered body weight or food intake59,60. Fto also affects

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Oxidative demethylationA chemical reaction in which the CH bond of a methyl group attached to a nitrogen or an oxygen atom is oxidized to OH by demethylases, and the intermediate decomposes to release the methyl group as formaldehyde.

development: Fto-knockout mice shows increased post-natal lethality and growth retardation59, and a homozy-gous loss-of-function mutation (Arg316Gln) in the FTO protein in humans leads to postnatal retardation, as well as multiple dysmorphisms and malformations61.

FTO is a member of the Fe(ii) and -ketoglutarate-dependent AlkB family of proteins that catalyse oxidative demethylation58; close homologues participate in epigenetic regulation, such as oxidative DNA demeth-ylation6264 and histone demethylation8. FTO was origi-nally shown to demethylate N3-methylthymidine in single-stranded DNA58 and N3-methyluridine in single-stranded RNA65 invitro; however, the function of FTO invivo remained unknown until we discovered that FTO efficiently demethylates m6A in both RNA and DNA invitro15. Further experiments showed that silenc-ing of FTO in HeLa and 293FT cells increased total m6A levels in polyadenylated RNA, and overexpression of FTO decreased m6A levels on RNA15. FTO is expressed in dot-like patterns in the nucleoplasm and partially colo-calizes with nuclear speckles. These cell-based results, together with observations that most mammalian cells and tissues contain very low levels (a few parts per million) of m6A on genomic DNA, led us to conclude that m6A on nuclear RNA (including mRNA, lncRNA and pos-sibly other types of RNA) is the main substrate of FTO. Recent work showed that m6A on three mRNA species could be demethylated by FTO invivo, and this function seems to affect neuronal activities66. FTO may also act as a nutrient sensor, which could modulate its demeth-ylation activities67. It should be noted that although FTO works preferentially on single-stranded RNA and DNA, it can still exhibit demethylation activity, albeit low, towards double-stranded RNA andDNA15.

The crystal structure of the FTO protein reveals an active domain that is similar to those of other pro-teins of the AlkB family68 (FIG.3c). FTO also contains a C-terminal domain with a novel fold that is distinct from other proteins of this family. This C-terminal domain may engage in additional proteinprotein or proteinRNA interactions to affect the function of FTO. The discovery of FTO as an m6A demethylase strongly sug-gests functional roles for m6A in human developmental regulation; however, to achieve the end goal of uncover-ing the underlying mechanism, a considerable amount of future work is required to identify the physiological RNA targets of FTO and to elucidate the functional consequences of such demethylation.

Demethylation of m6A in RNA by ALKBH5. ALKBH5 is another protein of the AlkB family that shows effi-cient demethylation activity towards m6A in mRNA and other types of nuclear RNA16,69. ALKBH5 has an alanine-rich sequence and a potential coiled-coil struc-ture in its N-terminus (FIG.3c), which may be important for its localization. ALKBH5 knockdown in human cell lines led not only to increased total m6A levels on poly-adenylated RNA but also to accelerated export of these RNAs from the nucleus to the cytoplasm16. However, the underlying mechanism is not fully understood. ALKBH5 and its demethylation activity affect nascent

mRNA synthesis and the rate of splicing16. Unlike FTO, direct immunoprecipitation of ALKBH5 has identified bound RNA substrates16, and ALKBH5 has been shown to be part of the mRNA-bound proteome70, which sug-gests a tight interaction with mRNA and other RNA substrates. ALKBH5 also colocalizes well with nuclear speckles in an RNase A-sensitive manner. Alkbh5 has the highest expression level in mouse testes. Consistently, Alkbh5-knockout male mice exhibit aberrant spermato-genesis, which is probably a result of altered expression of spermatogenesis-related genes16.

hm6A and f6A modifications on mammalian mRNA. While investigating FTO-catalysed demethyla-tion, we observed two unprecedented intermedi-ates, N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (f 6A), which were generated through the FTO-catalysed oxidation of m6A (REF.71) (BOX 1;FIG.3d). The hm6A intermediate is a direct oxi-dation product of m6A, and f 6A is the further oxidized product of hm6A. Both hm6A and f 6A can decompose in water to yield unmethylated adenine and formaldehyde (from hm6A) or formic acid (from f6A). To our surprise, these modifications are metastable under physiological conditions in neutral buffered solutions at 37 C with half-lives of ~3hours. This observation raised the pos-sibility that both modifications could exist in living cells and could have functional implications, given that median mammalian RNA half-lives are ~5hours72,73. Indeed, using a modified protocol to avoid acid, base and heating treatments, we have detected the presence of these modifications in mRNA isolated from mouse tissue and human cell lines71. The exact sources of these modifications invivo remain unknown so far; however, these new modifications carry functional groups that are distinct from m6A and could substantially affect RNAprotein interactions.

Although both FTO and ALKBH5 are mainly found in the nucleus, the possibility that both proteins could translocate to the cytoplasm under certain circumstances should not be ruled out. Cytoplasmic RNA may also be demethylated by these enzymes or by other currently unknown demethylases. ALKBH5 is only conserved in vertebrates from fish to humans, whereas FTO is con-served in vertebrates and has homologues in marine algae74. As there are many Fe(ii) and -ketoglutarate-dependent dioxygenases with unknown functions in var-ious organisms75, we should not be surprised to see the discovery of more m6A demethylases. In addition to its occurrence in eukaryotic mRNA, m6A also exists in vari-ous classes of RNA in eukaryotes, bacteria and archaea, including rRNA, tRNA and snRNA14. Furthermore, chemical modifications can also occur on various nitro-gen, carbon and oxygen atoms within the bases and backbone of RNA13 (BOX 1). These modifications (for example, methylation) on the heteroatoms oxygen and nitrogen can, in principle, be enzymatically reversed through the oxidative demethylation mechanism used by FTO and ALKBH5 or through nucleophilic substitu-tions (BOX 1). Demethylases that remove these other RNA methylations could exist and exhibit functionalroles.

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m6A reader proteins and effector functionsThe discoveries of m6A RNA demethylation and demethylases validate our hypothesis that the ubiqui-tous m6A modification is dynamic and reversible, which is similar to epigenetic DNA and histone modifications. The noticeable phenotypes of both FTO and Alkbh5 mutations in humans and mice strongly indicate the functional importance of this reversible m6A methyla-tion on RNA. For the m6A group to have a biological function, it needs to be recognized through reading by specific proteins. This process could resemble the roles of proteins that read 5-methylcytosine (m5C; also known as 5mC) in DNA, or methylated or acetylated amino acid residues of histones in order to exhibit the biological function associated with the modifica-tions and to enable reversible tuning. We can envision three types of selective reading mechanisms for m6A on RNA: first, a reader protein could selectively bind to the

m6A-containing RNA; second, the presence of m6A in a specific sequence could weaken the cognate binding interaction of an RNA-binding protein; and third, the presence of m6A may change the secondary structures of RNA and therefore alter proteinRNA interactions.

YTHDF2 preferentially recognizes m6A-containing mRNA and regulates both mRNA stability and localiza-tion. Using pulldown experiments, we have identified three cytoplasmic proteins of the YTH domain fam-ily, YTHDF13, as selective m6A-binding proteins in mammalian cell extracts17,76 (FIG. 4a). The YTH domain family consists of abundant RNA-binding proteins that previously had no clear function assigned. We con-firmed that mammalian YTHDF proteins preferentially bind to RNA that contains m6A at the G[G>A]m6ACU consensus sequence relative to unmethylated RNA of the same sequence76. Additionally, RNA probes that

Box 1 | RNA modifications


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PhosphateRiboseRiboseRiboseRibose

RNA backboneUCGA

a

b

c

Base

Oxidation

Oxidation

+

HC

N

H

NC

OH(Formic acid)(e.g. Adenine)(e.g. f6A)

OH+

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O

(Formaldyhyde)

(e.g. Adenine)(e.g. hm6A)(e.g. m

6A)

Cellular RNA species contain more than 100 chemical modifications with diverse properties. Chemical modifications of RNA can occur on the N1, N3, N7 and C8 atoms in both adenine and guanine; C2 and N6 in adenine; N2 and O6 in guanine; N1, O2, N3 and C5 in cytosine and uracil; N4 in cytosine and O4 in uracil; as well as on 2O of the ribose backbone and the OH group of the phosphate backbone (see the figure, part a). These modifications can modulate hydrophobicity, steric and electrostatic effects, and hydrogenbonding abilities of RNA bases and backbones. Methylation or other forms of alkylation on nitrogen or oxygen atoms can be removed through either an oxidative or a nucleophilic substitution mechanism. The oxidative demethylation (see the figure, part b) is best exemplified by Fe(ii) and ketoglutaratedependent dioxygenase enzymes, which use Fe(ii) as a catalytic centre, O

2 as an oxidant and

ketoglutarate as a cofactor. When the methyl group is linked to a heteroatom such as nitrogen or oxygen, the oxidation of CH to a hemiaminal or hemiacetal intermediate destabilizes the CN or CO bond, respectively, which leads to the demethylated product with the release of formaldehyde. The hemiaminal intermediate, such as N6hydroxymethyladenosine (hm6A), may undergo further oxidation to produce a formamide, such as N6formyladenosine (f6A), which can decompose in water to yield the demethylated product with the release of formic acid. The demethylation activity could be modulated by the effective concentrations of Fe(ii), O

2 or ketoglutarate. The bimolecular

nucleophilic substitution (Sn2) mechanism could also be used to remove RNA methylation on heteroatoms; however, such a process has yet to be shown for RNA demethylation (see the figure, part c).

m6A, N6methyladenosine; Nu, nucleophile.

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Ribosome profilingQualitative and quantitative sequencing of the RNA attached to ribosomes as a signature of genes that are expressed.

Processing bodies(P-bodies). Distinct foci in the cytoplasm that are enriched with RNA degradation factors.

contain adenine, hm6A or f 6A, or that have m6A in non-consensus sequences have decreased binding affinity. The enrichment of m6A in RNA immunoprecipitated with YTHDF13 further supports the role of YTHDF proteins as m6A-specific RNA-binding proteins.

RNA immunoprecipitation and PARCLIP experi-ments revealed mostly mRNA as targets of YTHDF2, in addition to some lncRNA targets. The binding sites localize around stop codons and at 3UTRs with a

conserved GAC[U>A] motif; thus, the occupancy of YTHDF2 resembles the distribution pattern of m6A on mRNA. Notably, the knockdown of YTHDF2 led to decreased half-lives of these RNA targets but had minor effects on the mRNA levels in the actively translating pool. Ribosome profiling further suggests that YTHDF2 alters ribosome occupancy of its mRNA targets. These results suggest that YTHDF2 has a role in RNA decay. Fluorescence immunostaining of YTHDF2 and fluores-cence insitu hybridization of its cognate mRNA revealed that YTHDF2 binds to m6A through the C-terminal YTH domain and localizes the cognate mRNA to processing bodies (P-bodies) for accelerated degradation through its N-terminal Pro/Gln/Asn-rich domain (FIG. 4). The exact RNA degradation mechanism needs to be further elucidated; however, YTHDF2 binds to mRNA with shorter poly(A) tails and does not seem to affect the deadenylation process76.

Several cytoplasmic mRNA decay pathways are known7786. The YTHDF2-mediated mRNA degrada-tion, which affects thousands of mRNA molecules, is a unique process that is dependent on the methylation of the target mRNA and could therefore be reversibly tuned through m6A methylation and demethylation. This discovery, together with the negative correlation of m6A with mRNA stability in general as revealed by knockdown of methyltransferases40, suggests one main function of m6A on RNA: the regulated degradation of methylated RNA. This process is mediated through selective m6A recognition and subsequent relocaliza-tion by a reader or effector protein. The control of the stability of the non-translating pool of mRNA (or other RNA species) through the YTHDF2-dependent mecha-nism could be important under various circumstances for the selective elimination of a group of RNAs77. Interestingly, Mmi1 the homologue of YTHDF pro-teins in Schizosaccharomyces pombe (FIG.4a) is essen-tial for the elimination of meiosis-specific transcripts during meiosis87. However, the presence of m6A has not been reported in S.pombe, which lacks homologues of METTL3 and METTL14. The potential presence of m6A in mRNA and its functional roles in S.pombe should be further investigated.

hnRNPs could be potential nuclear m6A readers. Besides the YTH domain family of proteins and other cytoplas-mic mRNA-binding proteins, pulldown experiments have also identified proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) type as potential m6A-selective binding proteins17. Known to form ribo-nucleoprotein granules that could affect mRNA localiza-tion and transport, hnRNPs could also block binding of splicing factors and affect alternative splicing. Additional experiments are required to investigate connections between hnRNPs andm6A.

Anti-readers of m6A and m6A-derived modifications. The presence of the methyl group can also disfavour binding of an RNA-binding protein to the modified RNA. This mechanism of anti-reading has yet to be observed for m6A. The m6A modification is widely


?

?

Translation

Degradation

RNA

Localization(strorage or transport)

YTHDF2

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f di

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06 183 9 12 15

mRNA lifetime (hours)

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S. cerevisiae Mrb1

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Human YTHDF1

Human YTHDF2

Human YTHDF3 YTH domain

YTH domain

YTH domain

P/Q/N-rich

P/Q/N-rich

P/Q/N-rich

YTH domain

YTH domain

Non-m6Am6A

Anm7Gppp

DNA

Transcription

YTH

P/Q/N-rich

MeAnm7Gppp

Me

Figure 4 | Functions of the reader (that is, effector) proteins of m6A. a | The characterized YTHDF proteins serve as N6-methyladenosine (m6A) readers. Human YTHDF13 proteins contain a carboxy-terminal YTH RNA-binding domain and an aminoterminal P/Q/Nrich region. The YTH domain protein is conserved in the fission yeast Schizosaccharomyces pombe and the budding yeast Saccharomyces cerevisiae. b | The m6A modification is enriched in mRNAs with shorter half-lives in general, which supports the proposed main role of m6A in regulating mRNA stability. c | The m6A-specific RNA-binding proteins are engaged in post-transcriptional regulation of gene expression. YTHDF2 regulates the methylation (me)-dependent RNA degradation. Other reader proteins may exist and affect RNA splicing, storage, trafficking and translation. Data in part b courtesy of X.Wang, laboratory of C.H.

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distributed in the 3UTRs of mRNA transcripts a region bound by numerous RNA-binding proteins that regulate mRNA metabolism and translation. It is possi-ble that certain anti-reading mechanisms exist to regu-late the fate of methylated mRNA. m6A is also known to protect RNA from recognition by cellular innate immu-nity proteins. Toll-like receptor 3 (TLR3) and TLR7 rec-ognize unmodified double-stranded and single-stranded RNA as invasive RNA species and selectively target them for degradation88,89. The incorporation of m6A and other RNA modifications in transfected exogenous RNA can reduce the recognition by innate immune systems to prevent unnecessary degradation, which increases their expression. An anti-reading mechanism possibly operates in this process.

Indirect reading. Certain RNA modifications, such as pseudouridine (), are known to cause secondary and tertiary structural changes90. The m6A modification reduces the base-pairing energy of A:U only margin-ally35, but this difference may shift the equilibrium of certain secondary and tertiary structures of RNA. The altered structures could have an effect on the binding of specific proteins, leading to indirect reading and regulation. In a recent study of HuR (also known as ELAVL1) a well-known RNA-binding protein that affects the stability of many mRNA transcripts in mam-malian cells9196 the m6A modification affected the ability of HuR to bind to different RNA probes invitro42. In this particular case, the RNA structure altered by methylation might indirectly contribute to the acces-sibility of the cognate HuR-binding site. However, the consensus sequence recognized by HuR invivo is different from the m6A-containing sequence91,92. The extent and details of the cellular connection between HuR and m6A still need to be further investigated. So far, no cellular example is known for this indirect reading mechanism.

Biological consequences of m6AThe recent breakthroughs in the discovery and charac-terization of m6A writers, erasers and readers, together with the parallel development of high-throughput assays that profile this methylation on a transcriptome-wide scale, set the stage and provide tools for functional inves-tigations that aim to identify the mechanisms by which m6A is translated into biological outcomes. Past studies that used broad-spectrum methylation inhibitors have yielded inconclusive results. Now that writer, eraser and certain reader proteins have been clearly defined, per-turbation of these machineries can lead to more specific phenotypic outcomes and experimental observations that will help to elucidate the biological roles of m6A and the underlying mechanisms. An instrumental aspect of this endeavour will be to categorize the phenotypic lev-els influenced by m6A. First are effects at the levels of whole organisms or tissues; studies at these levels could reveal tissue specificity of m6A, as well as its relevance to certain diseases and biological processes (for exam-ple, development, infertility, carcinogenesis, stemness, meiosis and circadian rhythm). Second are effects at the

pathway level (for example, the p53-mediated pathway, Notch signalling, nutrient sensing through mammalian target of rapamycin complex1 (mTORC1) and apopto-sis). Third are roles at the machinery level (for example, the spliceosome and the nuclear export machinery). Last are functions at the elementary molecular level on which all other levels depend (for example, thermodynamics and proteinm6A interactions). The reader proteins and their associated recognition mechanisms will be crucial in revealing and understanding theseroles.

Post-transcriptional regulation through methylation-dependent localization of the target transcript. To our knowledge, the in-depth characterization of YTHDF2 as the first m6A reader delineates the first established molecular pathway mediated by m6A: the binding of YTHDF2 to thousands of mRNA transcripts (and also to certain ncRNA transcripts) results in the localization of bound mRNA from the translatable pool to decay sites, thereby affecting the translation status and half-life of mRNA76. This discovery has two fundamental merits. First, it indicates that a main function of m6A methylation as a reversible mark is to affect mRNA stability, which fits nicely with the negative correla-tion between m6A levels and transcript abundance observed upon silencing of methyltransferases. In fact, such methylation generally associates with mRNAs that have shorter half-lives (FIG.4b), which further supports this notion. Second, this example illustrates how selec-tive reading of the m6A mark by a binding protein can affect localization of the target RNA, thus providing a model that applies to other potential readers which may broadly affect RNA transport, storage, stability, translation and splicing.

Although transcriptional regulation has major roles in regulating gene expression, it is protein levels that mainly determine biological phenotypes. Protein production is also subjected to various types of post-transcriptional regulation such as through mRNA secondary structure, microRNAs or mRNA translational control which probably contributes as much as, if not more than, transcriptional regulation to determine cel-lular protein abundance97103. m6A methylation provides a new dimension of post-transcriptional gene regulation. The m6A mark on mRNA could affect the abundance, localization and use of mRNA, and potentially splicing; all of these represent central processes that are connected to protein expression (FIG.4c). We believe that m6A has a substantial contribution to the post-transcriptional balance that regulates proteinlevels.

Known examples of RNA methylation in regulating cel-lular processes. Various circadian RNAs and clock output transcripts contain the m6A modification104. Inhibition of m6A formation leads to prolonged nuclear reten-tion of circadian RNAs and thus delays the nuclear exit of mature period circadian clock 2 (Per2) and aryl hydrocarbon receptor nuclear translocator-like (Arntl; also known as Bmal1) mRNAs104. This observation is consistent with our discovery that deletion of ALKBH5 (which increases m6A levels) in mammalian cells leads

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to reduced nuclear retention time16, although it is not clear whether m6A has a direct cis role in modulating the export specifically of m6A-containing RNA molecules or whether it is an indirect consequence of perturbation to the RNA export machinery.

The m6A RNA modification is involved in priming yeast cells to bipotential states and meiosis during nitro-gen starvation. Through carefully monitoring methyla-tion profiles at different stages, a recent study suggests that methylation is important for the kinetic control of RNAs during the meiotic prophase32. Although no marked change in half-lives has been observed for the m6A-containing RNAs, the accessibility of these RNAs to translation may be modulated through interac-tions with potential reader proteins. Analogous to the proposed function of m6A in accelerating both RNA export and degradation in mammalian cells, m6A may ensure faster turnover of the RNA transcripts that are important during the meiotic prophase but that are harmful and need to be degraded after the exit from prophase. This example suggests that m6A could glob-ally ensure fast kinetic responses by redirecting RNA to different organelles and by quickly decreasing the expression of related proteins. A similar mechanism could also affect eukaryotic mitosis.

Similarly, another study in mESCs revealed that the m6A methylation accelerates transcript decay, which is consistent with the main role that we propose for m6A, and affects stem cell maintenance and differen-tiation42. Interestingly, when compared with pluripo-tency-related genes, developmental regulators were significantly enriched among target genes of METTL3 and METTL14 in mESCs. In particular, m6A destabi-lizes developmental regulator transcripts, which may suggest that methylation is important for maintenance and differentiation of mESCs. Temporal and spatial regulation of mRNA is known to have crucial roles in embryonic development. Post-fertilization, maternal mRNA needs to be degraded in a programmed manner. The methylation on mRNA could affect this process through altering the localization and half-lives of tar-get mRNA transcripts. Such methylation could mark specific sets of RNA species and therefore differentiate between maternal and zygotic mRNA in a kinetic man-ner. Heritable information could perhaps be passed down to generations of cells through orchestrated RNA methylation and demethylation activities.

Advantage and specificity of the m6A-based regula-tion. The first m6A reader protein to be characterized is known to affect more than 3,000 different mRNA tran-scripts76. We propose that the reversible RNA methyla-tion pathway, in general, has evolved to affect processes that involve changes in the expression of large groups of genes. This property is intimately related to the poten-tial advantages of reversible methylation at the RNA level. Besides providing increased complexity to the regulatory network, this mechanism may allow rapid responses to signalling and stimuli when the expres-sion of a group of proteins (which can range from tens to thousands) needs to be adjusted in a rapid manner;

when the response at the DNA level (that is, transcrip-tion) could be too slow; and when the response at the protein level may require specific interactions or modifications to tens to thousands of proteins, which is difficult to achieve. Reversible methylation or other forms of modifications on mRNA provides the best option. A specific sequence that can undergo revers-ible modification, and thus be subjected to regulation, can be readily included in a group of mRNA transcripts (for example, at their 3UTRs) and lncRNAs in order to affect RNA stability, localization and translatability, as shown in the example ofYTHDF2.

PerspectivesIn summary, reversible RNA methylation shares many of the same characteristics as epigenetic DNA and his-tone modifications. Expression levels and potentially post-translational modifications of writers, erasers and readers can constantly sculpt the RNA methyl-ome (FIG.5), which might in turn affect the eventual protein expression. Therefore, the reversible chemical tagging that dynamically controls the outcome of gene expression occurs in all three main components of the central dogma. Whereas epigenetic DNA and histone modifications affect mostly transcriptional events, reversible RNA methylation mainly has an impact on regulation of post-transcriptional gene expression and could directly affect protein production. Indeed, recent research indicates that cellular protein levels are not necessarily correlated with the mRNA levels105,106, which emphasizes the importance of post-transcriptional regulation of gene expression. Owing to the shared use of reversible chemical tagging for dynamic gene expression control, reversible RNA methylation has been compared with epigenetic DNA and histone modifications10,107. To also be a true epigenetic mark, m6A would need to be heritable through cell division; although this has not yet been demonstrated, such

Figure 5 | RNA methylation could affect various aspects of RNA metabolism and mRNA translation, and regulate protein expression post-transcrptionally. Whereas N6-methyladenosine (m6A) methyltransferases and demethylases shape the methylation (me) landscape, the reader proteins bind to the methylated RNA and mediate specific functions. Various cellular processes could be affected by m6A RNA methylation. In the cell nucleus, m6A may affect RNA export, nuclear retention and splicing, possibly through interactions of reader proteins with RNA export, retention and splicing machineries. After RNAs are exported to the cytoplasm, YTHDF2 can bind to the m6A-containing RNAs and direct them to processing bodies (Pbodies) for accelerated mRNA decay. Pbodies can dynamically form stress granules, in which RNAs could be stored and released back to the translating pool. Besides YTHDF2, other m6A reader proteins may bind to m6A-containing RNAs to control their transport and storage, thereby affecting translation. FTO, -ketoglutarate-dependent dioxygenase FTO; WTAP, Wilms tumour 1-associating protein.

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Anm7Gppp

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METTL14 METTL3

WTAP

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RNA polymerase II

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a possibility is conceivable through direct passage of writer, reader and eraser proteins or methylated RNA between generations of cells, which is a research direction that needs to be further explored.

Many challenges lie ahead. It will be important to clearly define the spatiotemporal properties of m6A in terms of tissue specificity and in response to exter-nal and internal cues. The top priority is to elucidate the involvement of m6A in RNA degradation, trans-port, storage, translation and splicing. The first steps to achieve this goal will involve identifying and elu-cidating the functions of m6A reader proteins. Some additional questions are: what is the interplay between methyltransferases and demethylases that orchestrate the methylation status of individual sites? How is methylation and demethylation selectivity achieved? Is the methylation coupled with transcription, and do the two processes have mutual interactions? From yeast to humans, how do the functions of m6A relate to cell phenotypes and cell behaviour? Could some of the processes be targeted to regulate biological functions or to treat human diseases? Further research will answer some of these questions and reveal fundamental aspects of m6A biology.

Other intriguing chemical modifications exist on mRNA and other types of nuclear RNA, such as m5C, and 2-OMe. Some of these modifications are only a few fold less abundant than m6A on mRNA. They could also be dynamic and may have important roles in gene expression regulation, as recently suggested for 108110. Although transcriptome-wide m5C distribution has been mapped111113, the other two modifications have yet to be studied using modern sequencing approaches. Both and 2-OMe may have connections to human diseases, which suggests functional roles114116. Modifications on tRNA and rRNA can also be dynamic and could affect the outcome of protein expression. Of the nine human homologues of RNA demethylases, ALKBH2 and ALKBH3 are DNA repair enzymes that use the same oxidative demethylation mechanism to remove DNA methyl adducts117,118, and ALKBH8 is a tRNA hydroxylase119,120 that seems to affect tRNA codon usage, whereas ALKBH1, ALKBH4, ALKBH6 and ALKBH7 still do not have clearly defined functions. Some of these homologues might work on nucleic acids and act as demethylases for other forms of nucleic acid methylations. We are still at the very beginning of this new realm of fundamental research.

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AcknowledgementsThe authors apologize to colleagues whose work was not cited owing to space limitation. They thank T. Pan, X. Wang, Y. Yue and J. Liu for discussion. C.H. is supported by the US National Institutes of Health grants GM071440 and the EUREKA grant GM088599. This work was also supported partly by grants from the Israel Science Foundation, the Flight Attendant Medical Research Institute (FAMRI) and the Israeli Centers of Research Excellence. S.F. Reichard contributed to editing of this manuscript.

Competing interests statementThe authors declare no competing interests.

FURTHER INFORMATIONModomics a database of RNA modification pathways: http://modomics.genesilico.pl/Three-dimensional ribosomal modification maps database: http://people.biochem.umass.edu/fournierlab/3dmodmap/main.phptRNAmod prediction of tRNA modifications: http://crdd.osdd.net/raghava/trnamod/

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Abstract | Cellular RNAs carry diverse chemical modifications that used to be regarded as static and having minor roles in fine-tuning structural and functional properties of RNAs. In this Review, we focus on reversible methylation through the most prem6A RNA methylation in eukaryotesFigure 1 | Reversible chemical modifications that regulate the flow of genetic information.In the central dogma, genetic information is passed from DNA to RNA and then to protein. Epigenetic DNA modifications (for example, the formation of 5methylcytosiFigure 2 | Profiling of m6A in RNA by m6A RNA immunoprecipitation.Antibody-based N6-methyladenosine (m6A) RNA immunoprecipitation has been developed to profile the transcriptome-wide distribution of m6A. a | Isolated mRNA is fragmented to ~100nucleotidm6A writers in eukaryotesFigure 3 | Reversible m6A methylation of mRNA and other types of nuclear RNA.The N6-methyladenosine (m6A) modification is installed by a hetero complex of two methyltransferases METTL3METTL14, assisted by Wilms tumour1associating protein (WTAP), and m6A erasers in mammalsBox 1 | RNA modificationsm6A reader proteins and effector functionsFigure 4 | Functions of the reader (that is, effector) proteins of m6A.a | The characterized YTHDF proteins serve as N6-methyladenosine (m6A) readers. Human YTHDF13 proteins contain a carboxyterminal YTH RNA-binding domain and an aminoterminal P/Q/NBiological consequences of m6AFigure 5 | RNA methylation could affect various aspects of RNA metabolism and mRNA translation, and regulate protein expression post-transcrptionally.Whereas N6-methyladenosine (m6A) methyltransferases and demethylases shape the methylation (me) landscaPerspectives

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