Briefings in Functional Genomics Advance Access published October 10, 2014

B RIEFINGS IN FUNC TIONAL GENOMICS . page 1 of 11

doi:10.1093/bfgp/elu039

Role of the N6 -methyladenosine RNA mark in gene regulation and its implications on development and disease Udita Chandola, Radhika Das and Binay Panda

Epigenetics is a field that encompasses chemical modifications of DNA and histone proteins, both of which alter gene expression without changing the underlying nucleotide sequence. DNA methylation and modifications of histone tails have been studied in detail and are now known to be global gene regulatory mechanisms. An analogous post-transcriptional modification is chemical modification of specific nucleotides in RNA. Study of RNA modifications is a nascent field as yet, and the significance of these marks in controlling cell growth and differentiation is just beginning to be appreciated. The addition of a methyl group to adenosine (N-methyl- 6 -adenosine) or m6A is the most abundant modification in mammalian mRNAs. Though identified four decades ago, interest in this particular modification was set off by the discovery that the obesity gene FTO was an RNA demethylase. Since then, many studies have investigated m6A modification in different species. In this review, we summarize the current literature and hypotheses about the presence and function of this ubiquitous RNA modification in mammals, viruses, yeast and plants in terms of the consensus sequence and the methyltransferase/demethylation machinery identified thus far. We discuss its potential role in regulating molecular and physiological processes in each of these organisms, especially its role in RNA splicing, RNA degradation and development. We also enlist the methodologies developed so far, both locus-specific and transcriptome-wide, to study this modification. Lastly, we discuss whether m6A alterations have consequences on modulating disease aetiology, and speculate about its potential role in cancer. Keywords: m6A; RNA methylation; post-transcriptional regulation; METTL3; FTO

INTRODUCTION In the past two decades, research on epigenetic alterations in human diseases has been largely focused on DNA and histone modification events. These alterations play important roles in gene regulation, chromatin modifications and genome integrity. DNA methylation at CpG di-nucleotides in promoters, untranslated regions and first exons are widely studied epigenetic modifications in human diseases like cancer [1]. In addition to DNA methylation, methylation of

histones at lysine residues, along with other post-translational modifications like acetylation, phosphorylation, ubiquitination and sumoylation, also play important roles in gene expression and maintenance of chromatin structure [2]. Although presence of methylated regions has been described in RNA nearly 40 years ago [3, 4], alongside their possible roles in RNA processing in mammalian transcripts, the exact mechanism(s) through which the methylated RNAs act and control cellular processes are largely unexplored.

Corresponding author. Binay Panda, Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology, Bangalore, India. Tel.: þ91-80-28528900; Fax: þ91-80-28528904; E-mail: [email protected] Udita Chandola is a Junior Research Fellow at Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology. Radhika Das is a Project Scientist at Ganit Labs, Bio-IT Centre, Institute of Bioinformatics and Applied Biotechnology. Binay Panda is the Principal Investigator and heads Ganit Labs, Bio-IT Centre of the Institute of Bioinformatics and Applied Biotechnology, India. Ganit Labs, Bio-IT Centre is an initiative of Institute of Bioinformatics and Applied Biotechnology and Strand Life Sciences. ß The Author 2014. Published by Oxford University Press. All rights reserved. For permissions, please email: [email protected]

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Abstract

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m6A RNA METHYLATION IN DIFFERENT SPECIES Humans and mice The most common modification of mRNA in mammalian systems is m6A. Nearly 7600 mRNA transcripts and >300 non-coding RNAs contain the m6A mark in humans. These marks exhibit tissue-specific regulation with increased expression in liver, kidney and brain (the highest levels being present in the adult brain) [19]. Among the transcripts with the highest levels of m6A enrichment [7, 19], many have important biological functions in transcriptional regulation (FOXE3), intracellular signaling (CDKN2C) and neurological development (Bdnf, Dscam, Lis1 and Ube3a). The non-coding RNAs bearing the m6A mark are usually >200 nucleotides in length and include many of the conserved linc RNAs, which are critical for

Figure 1: m6A modification on RNA.

transcriptional regulation (examples being ‘X inactivation specific transcript’ and ‘Hox transcript antisense RNA’) [7, 20]. In humans and mice, m6A sites are usually present near stop codons and in 30 UTRs, within the consensus sequence of G(m6A)C (in 70% cases) or A(m6A)C (in the rest 30% cases) [19]. In both these species, m6A is posttranscriptionally added by the enzyme m6A methyltransferase METTL3 (MT-A70) [21]. The loss of MT-A70 results in apoptosis of human HeLa cells [19]. More recently, another component of the m6A methyltransferase machinery—METTL14— has been described in mouse embryonic stem cells, and is believed to act synergistically with METTL3 [22]. Wilm’s tumour-associated protein (WTAP) has also been shown to be a part of this machinery that recruits methyltransferase like 3 (METTL3)/ METTL14 to the RNA for methylation [23]. Moreover, two m6A demethylases in human— FTO [24] (fat mass and obesity-associated protein) and ALKBH5 [25] have been identified indicating the reversible nature of RNA methylation process in humans. The function of RNA methylation has been best studied in higher eukaryotes. It is believed to have a role in splicing because the RNA methylation machinery co-localizes with the nuclear speckles where splicing occurs [7]. However, more recently it was demonstrated that m6A selectively binds the human YTH domain family 2 (YTHDF2) proteins [26] and thus might help in regulation of mRNA degradation. The YTHDF2 protein binds to singlestranded RNA and has co-evolved in many eukaryotes. The YTHDF2 protein targets the bound mRNA to mRNA decay sites, such as processing bodies (P-bodies). In another study in mouse embryonic stem (ES) cells [22], Wang and co-workers proposed that m6A methylation leads to the degradation of RNA by the miRNA pathway rather than by binding of YTHDF2 proteins. They also

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Owing to the unstable nature of RNA, studies on transcriptome have been generally arduous. Despite this, more than a hundred types of nucleotide modifications have been identified in different RNA molecules, namely, messenger RNA (mRNA), long non-coding RNA (lncRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNA (snRNA) and some small nucleolar RNA (snoRNA) [5, 6]. Two types of RNA modifications have been widely reported in eukaryotic mRNA, N6-methyladenosine (m6A) (Figure 1) and 5-methylcytosine (m5C) that occur at the 30 untranslated region (30 UTR) and stop codon sites [7], suggesting their involvement in switching genes on/off and/or in facilitating the binding of protein(s) that mediate downstream biological function(s). A third type of modification in RNA, 5-hydroxymethyl cytosine (5hmC), was reported to be a signature for disease prognosis [8]. Modifications of nucleotides in general, both on DNA and RNA, and the use of chemical reagents to study modified nucleotides in RNA have been reviewed elsewhere [6, 9]. This review covers the m6A modification on mRNA, the most common modification in mRNA of mammalian systems, and the one that is believed to play an important role in biology and disease. Though originally described in tRNAs, rRNA and snoRNA [10–18], the ubiquitous distribution of the m6A modification in mRNA has generated the most interest owing to its potential to explain reversible gene regulation in cells.

Role of the m6A RNA mark

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showed that the m6A demethylation was associated with binding of the RNA stabilizing protein HuR [22] believed to maintain the ES cells in the ground state. The above results indicate that the dynamic m6A modification affects the translation status and lifetime of mRNA. Another study pointed out the role of m6A methylation in maintenance of the circadian rhythm of RNA expression [27]. Various enzymes and their roles in m6A RNA methylation are depicted in Figure 2.

Virus m6A has been detected in viral mRNA and in the RNA of retroviruses as well. It occurs with a non-random distribution in the sequences Gm6AC, Am6AC and PuGm6ACU [28]. The best studied virus in this regard is the Rous sarcoma virus (RSV), which has an average of 10–15 m6A residues per virion RNA molecule and is heterogeneous, i.e. different molecules of RNA showing different levels of methylation [28]. Nonetheless, in RSV, all the m6A is present only at the 30 ends of the RNA [28]. In contrast, Simian virus 40 (SV40) late mRNAs exhibit an average of three m6As per mRNA, all near their 50 ends [29]. Adenovirus

mRNAs are also found to contain m6A in the first two-thirds of their RNA [30, 31]. Stoltzfus and Dane [32] treated RSV-infected chicken embryo fibroblasts with cycloleucine to inhibit internal methylation and observed an increased level of unspliced viral RNA and reduced levels of spliced RNAs, thus indicating the role of m6A in splicing. In SV40-infected cells, Finkel and Groner [33] found that cycloleucine treatment led to an increase in polyadenylated RNA in the nucleus, but decrease in cytoplasmic mRNA, indicating a role of m6A in mRNA transport from nucleus to cytoplasm. Although the precise function of m6A in viral transcripts is unknown, it is believed to play a role in regulating mRNA processing [32, 33]. Moreover, the enzymes involved in RNA methylation and demethylation are yet to be elucidated.

Yeast The core RNA methyltransferase Mum2, Ime2 and Slz1 (MIS) complex has been identified in yeast, which is induced specifically during meiosis in Saccharomyces cerevisiae [34]. m6A methylation occurs at an RGAC (R ¼ A/G) core consensus sequence at the 30 ends of mRNAs, near the stop codon. MIS in

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Figure 2: Methylation and function of m6A RNA modification: RNA gets methylated with the help of enzymes METTL3/METTL14 and WTAP. The m6A modification in transcripts has a role in splicing and/or in mediating RNA instability where binding of YTHDF2 eventually leads the methylated RNA to P-bodies. However, under certain temporal and spatial conditions, the enzymes FTO/ALKBH5 directly undertake demethylation of the RNA at the same site, making the unmethylated RNA bound by the RNA stability factor HuR favouring constitutive expression.

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Plants (arabidopsis) m6A is ubiquitously present in Arabidopsis mRNA [41] and is predominantly positioned towards the 30 end of RNA transcripts (100–150 bp before the poly (A) tail). The METTL3 homolog MTA has been identified in Arabidopsis [41] and functions in laying down the m6A methylation marks on RNA. Reduced m6A levels cause decreased apical dominance, abnormal organ growth and an increased number of trichome branches in Arabidopsis [41]. Knockout studies of MTA have also been carried out to understand the function of m6A.MTA homozygous mutants are arrested at the globular stage of embryonic development, pointing towards the role of m6A in trichome branching [42]. Interestingly, overexpression of AtFIP37, a protein that interacts with MTA [42], leads to an increase in trichome branching [43]. As per the RNA modification database RNAMDB (http://mods.rna.albany.edu/), there

are about a hundred nucleotide modifications reported for RNA. The database provides a comprehensive listing of post-transcriptionally modified nucleosides in RNA. MODOMICS (http://modo mics.genesilico.pl/) and RNApathwaysDB (http:// www.genesilico.pl/rnapathwaysdb/) are two other complementary resources, which document RNA modifications and metabolism. While MODOMICS presents RNA modification pathways at the level of nucleosides, RNApathwaysDB deals with RNA metabolism with respect to whole RNA molecules. The integrated information from these databases and the experimental data accrued from various studies will provide insights for future investigations on m6A methylation.

Techniques for investigating m6A Methylation in RNA The detection of the presence and location of nucleoside modifications on RNA is pivotal for identification of genes that are altered and the conditions leading to such modifications. The earliest method entailed RNase cleavage in the presence of a complimentary oligodeoxyribonucleotide (protecting the area of interest), followed by mass spectroscopy [44, 45]. The advent of next-generation sequencing greatly facilitated identification of RNA modification sites in a high-throughput manner. An alternative method called methylated-RNA immunoprecipitation or Me-RIP [41, 46] was developed, which allowed whole-genome identification of m6A-modified RNA, by using an antibody against m6A to pull down fragmented RNA containing the m6A mark, followed by massively parallel sequencing [7, 41, 46]. In yeast, this method was further improved to lower the concentration of the input RNA [34]. Single-molecule real-time RNA sequencing and Site-specifc cleavage and radioactive-labeling followed by ligation-assisted extraction and thin layer chromatography (SCARLET) were subsequently developed to provide status of modified nucleotides (m6A) across the whole genome and specific site, respectively, at a single nucleotide resolution. It aided in locating and comparing m6A site status on lncRNA and mRNA, reporting false-positive m6A candidate sites identified previously and providing insight pertaining to the m6A-RNA structural motifs [47, 48]. A recently developed method, called High-Resolution Melting (HRM), provides a locus-specific high-resolution platform for

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yeast is made of three components: Ime4 (orthologous to mammalian METTL3), Mum2 [orthologous to mammalian Wilm’s-tumor-1-associated protein (WTAP)] and Slz1 [35]. Yeast mutants harbouring mutations in any of the three components are not lethal and show impaired meiosis [35–38], providing an advantage to study RNA methylation in yeast over the mammalian systems. Because no RNA demethylases have been identified in yeast, it has been suggested that reduction in m6A is probably coupled to MIS complex down-regulation [34]. However, several proteins, such as MRB1, containing the YTH RNA-binding domain, have been identified and are reported to be coevolved with mammalian m6A readers [39]. The Schizosaccharomyces pombe Mrb1ortholog, Mmi1p, is required for eliminating meiosis-specific transcripts during vegetative growth [40], suggesting a role for m6A methylation in meiosis. Hence, it appears that m6A plays a major role during meiosis in yeast. Moreover, unlike in other organisms where RNA methylation shows tissue and developmental-stage specific regulation, in yeast, m6A methylation levels vary over a meiotic time course. In S. cerevisiae, the sequence motif, 30 position and lack of local secondary structure act as the local (Cis- regulatory) factors and IME1 and NDT80 proteins act as global (trans- regulatory) factors for regulation of m6A methylation levels during the meiotic program [34].

Role of the m6A RNA mark direct detection of altered bases on RNA based on the melting property of nucleic acids, thus avoiding loss of information due to chemical/enzymatic distortion of the sequence [49]. Additionally, high-throughput sequencing cross-linking immunoprecipitation (HITS-CLIP) technique can be used to highlight the m6A sites on the mRNA post–ultraviolet (UV)-induced RNA-m6A antibody cross-linking and sequencing [20, 50, 51]. The techniques used for investigation of m6A RNA methylation are illustrated in Figure 3 and their applications and limitations are summarized in Table 1.

The role of RNA methylation in disease has not been studied directly, but the methylases and demethylases of m6A have been shown to have disease correlations. METTL3 (m6A methylase) is expressed in all human tissues with highest being in testis [54, 55]. Genes enriched in the METTL3 knockdown studies belong to the p53-signaling pathway and are involved in apoptosis [19]. WTAP was first recognized as a protein associated with Wilm’s tumor [23] but more recently has been found to be essential for cell cycle progression [23]. Knockdown of WTAP leads to a variation in RNA isoforms, suggesting its role in splicing [23]. In zebra fish embryos, knockdown of WTAP leads to embryonic defects, pointing towards its role in mammalian development [23]. FTO (m6A demethylase) is proposed to be a major obesity factor [24], discovered in many linkage studies (though a recent report suggests that its long-range association with IRX3 may be causative of obesity rather than FTO itself) [56]. FTO is a member of the ALKB family of enzymes, which catalyse oxidative demethylation of m6A. ALKBH5 is the other mammalian demethylase, other than FTO that oxidatively demethylates m6A in mRNA [25]. ALKBH5 is associated with certain mRNAprocessing factors in nuclear speckles and may thus affect mRNA export and metabolism [25]. ALKBH5 has the highest expression level in mice testes, and mice deficient in this showed spermatogenic maturation arrest resulting in impaired fertility [25]. ALKBH5-deficient mice show differential expression of genes involved in

spermatogenesis and in the p53 functional interaction network [25] pointing towards a global role for RNA methylation-mediated regulation in health and disease.

Implications on RNA structure and protein binding Many of the m6A sites are present in hairpin stems of RNA secondary structure [48, 57].These m6A residues are located adjacent to two consecutive non-canonical base pairs. It is hypothesized that this could result in opening of the major groove to facilitate the interaction of the m6A residue with binding proteins [19]. Although the nature of all the proteins that could bind to the more accessible sites is not known, the analyses of interacting proteins in the context of diseases should provide useful information.

IMPLICATION ON IMMUNOGENIC POTENTIAL Toll-like receptors (TLRs) are the most conserved molecules of the innate immune system that establish the first line of defense against besieging pathogens. They are a version of non-catalytic leucine-rich pattern recognition receptors, embedded in sentinel cells, which recognize pathogen-associated molecular patterns facilitating an immune response. They recognize lipopeptides, peptidoglycans, lipotechoic acid, zymosan, oligosaccharides, heat shock proteins and, more importantly, nucleic acids, specifically RNA. It is hypothesized that the hundred different nucleotide modifications might generate immunogenicity in the RNA through TLR recognition [58, 59]. These modifications provide an additional molecular feature for immune cells to discriminate between microbial and host (mammalian) RNA, leading to differential TLR signals generated in response to RNA. Naturally occurring unmodified RNA, which stimulate TLR3, TLR7 or TLR8 do not have the same potency as the modified RNAs [59]. Distinct TLRs respond differently to modified nucleosides. RNA with the m6A modification does not activate TLR3, and those with m5C and/or m6A do not activate TLR7 or TLR8 [59, 60]. The presence of the modified base pair may thus lead to non-recognition of the pathogen carrying these (e.g.: a viral nucleic acid) by the TLR receptor. These undetected viral components may then stimulate a pathway involved in cancer development [60]. TLR-mediated signaling

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CONSEQUENCES OF RNA METHYLATION Implications in disease

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Figure 3: Techniques used for detection and analysis of the m6A RNA modification. (A) Site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin layer chromatography (SCARLET). (B) me-RIP. (C) HRM curve analysis.

also produces several anti-cancer proteins, and thus this is often considered a target for cancer therapy [61]. Prominent examples in this category are TLR3 ligand (double-stranded RNA) or TLR3 agonists

(polyA-U and poly-C) that are used to stimulate apoptosis of breast and prostate cancer cells [60]). It is known that human papilloma virus has the ability to modulate TLR expression, leading to tumor

High UV cross-linking-induced irreversible covalent bond formation between m6A site in mRNA and the m6A binding protein, followed by cell lysis, immunoprecipitation, washing, proteinase K digestion, leaving only the m6A bound peptide, followed by adapter ligation and cDNA synthesis and sequencing to identify the protein ^RNA m6A crosssites. Low RNA-digestion followed by RNA fragmentation with mass spectroscopy [44, 45] RNaseH in the presence of complimentary oligodeoxyribose nucleotide pair, followed by purification and digestion with RNase A/RNase T1. The resulting oligonucleotide

HITS-CLIP [20, 50, 51]

Does not provide a single nucleotide resolution of the modification (resolution of 24 nt around the methylation sites), loss of isoform information during fragmentation and cDNA conversion and the enrichment step leads to a stoichiometric loss of information. The starting input RNA concentration is too high (300 mg). Loss of isoform and stoichiometric information due to fragmentation and immunoprecipitation, respectively. Stringent filtering criteria may result in false negatives and some false positives, thus affecting the nucleotide affinity assay.

Global view of transcriptome-wide mapping of m6A modifications.

Locus-specific detection of m6A on the RNA.

(continued)

The technique is laborious and is unable to scale up for genome-wide detection.

Global view of transcriptome-wide mapping of m6A modification at increased resolution (around 3 nt median distance between the enriched peak and nearest consensus sequence). This was achieved by decreasing the fragment size (from 100 to 50 nt) and using ligation-based strand-specific library preparation protocol capturing both the ends of the fragment, ensuring that the methylated base lies within the sequence fragment. Further, the false positive can be detected by using negative control, a strain, which does not accumulate m6A. Genome-wide RNA^ protein interactions and pos- Low efficiency and non-specificity of UV irradisible identification of RNA m6A target. It can be ation, unprecedented damage responses induced performed on most tissues. post-UV treatment, inefficient reverse transcription and sequencing of modified bases, lack of reproducible information across in vivo and in vitro experiments, inability of available bioinformatics tools to normalize tags in both exons and introns.

Limitations

Application

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Non-antibody based

An optimized version of Me-RIP- High seq technique.

Modified Me-RIP-seq [34]

High

m6A-antibody-mediated enrichment of methylated-RNA fragments followed by massively parallel sequencing.

Me-RIP-seqç Figure 3B [41, 46]

Throughput

Antibody based

Description

Name of technique

Method

Table 1: Techniques used for m6A RNA modification studies

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Method

SCARLET çFigure 3A [48]

Single-nucleotide-real-time sequencing [47].

HRMçFigure 3C [49]

Name of technique

Low

Throughput

Application

Site-specific and high-resolution detection of modified bases on rRNA, tRNA and snRNA by binding of oligonucleotide probes to the template total/purified RNA. The optimal concentration of 23 S rRNA for implementation of the method is 0.4 mM. It does not require PCR amplification, change in nucleotide sequence through chemical modifications or fragmentation, which may lead to a loss of information. The technique may offer detection of modifications on the rRNA that lead to antibiotic resistance [63]. In a single molecule/high-resolution and locus-speDetects the incorporation time Low cific technique, modified RNA is observed in or interpulse durations for a real time during cDNA conversion in presence of single phospholinked nucleospecific fluorescently labelled DNA primer. It tide by reverse transcriptase provides information about the relatively static enzyme by fluorescence, and stable secondary structures and the structherefore, aiding differentitural rearrangement in the RNA. ation between native and High (PacBIO) Genome-wide quantitative mapping of m6A resimodified bases. dues based on the kinetic variation data obtained during standard sequencing with Pacific Biosciences platform. It has been used in the past for detection of target sites for enzymes that catalyse such modifications and isoform types (Iso-seq) [52, 53]. Low Detection of m6A status (location and fraction) is Site-specific cleavage of m6A achieved in a candidate site/locus specific in containing candidate sites folmRNA/lncRNA at a single nucleotide resolution. lowed by radiolabelling, ligaThe entire RNA can be used, thus, eliminating tion and nuclease digestion. the purification step. It has been used to check Isolation of m6A containing the methylation status of sites that had earlier residue on the template been reported as methylated and also report through thin-layer chromatogseveral incomplete m6A-modified candidate reraphy is performed. gions [37]. It has been used to detect m6A sites in RNA secondary structures [37].

fragments are analysed by mass spectrometry. Detects nucleotide modifications in RNA/DNA based on the alteration in the melting curve of the duplex and due to the base pairing and stacking interaction using quantitative polymerase chain reaction (PCR).

Description

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Table 1: Continued

Requires laborious experimental procedures like radiolabelling, ligation, ribonuclease digestion and thin-layer chromatography. The technique cannot be scaled up for genome-wide detection.

Loading bias due to the slow diffusion of the long reads may occur.

The transcriptase activity across a specific RNA template maybe hampered by the bulky fluorescence probes attached to the base analogues.

Cannot detect nucleotide modifications that do not significantly alter the melting property of the nucleic acid duplex. It also requires partial enrichment of non-ribosomal targets by an additional purification step. The presence of an alternate modified base or a strong secondary structure in the middle of the RNA region complimentary to the oligonucleotide may prevent its hybridization.

Limitations

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Role of the m6A RNA mark progression [62]. Because several modified bases of RNA play a role in interfering/stimulating TLR signaling pathway [62, 64], it is possible that viralinduced RNA modification plays a role in the process of cancer progression.

Concluding remarks

Key Points  The N6 methyladenosine mark (m6A) is the most abundant nucleotide modification in eukaryotic mRNAs and other viral, yeast and plant RNA species.

 The consensus sequence for its addition in conserved: G/A (m6A)C and the sites are usually present at 30 UTRs or near stop codons.  The methyltransferase machinery consisting of METTL3/ METTL14 and WTAP facilitates addition of the m6A mark. Demethylation is catalysed by FTO and ALKBH5 in humans.  The exact function of the m6A mark is under scrutiny, but it has been implicated in RNA splicing, mRNA degradation and stability, cell differentiation as well as meiosis (in the case of yeast).  The role of m6A-RNA in basic cellular processes has been established by the knockout of the methylases/demethylases studies.  The role of m6A-RNA in diseases remains to be defined.  Addition of m6A might have a direct impact on RNA secondary structure and thus may affect protein binding, as well as affecting the immunogenic potential of the RNA species.  Newer generations of DNA sequencing techniques are greatly enhancing our ability to discover m6A modifications on RNA at a whole-genome level.

Acknowledgements The authors thank Dr Reinhard Brunmeir, Singapore Institute for Clinical Sciences, A*STAR, Singapore for critical review of the manuscript.

FUNDING Research at Ganit Labs, Bio-IT Centre is funded by grants from the Department of Biotechnology (DBT), Ref no: BT/01/ CEIB/11/IV/05, 22-08-13; the Department of Science and Technology (DST), Ref no: 100/IFD/C/1355/2014-2015; the Department of Electronics & Information Technology (DEITY), Ref no:18(4)/2010-E-Infra., 31-03-2010; the Council of Scientific and Industrial Research (CSIR), Ref no: MLP-901, 28-02-2013, all Government of India agencies; and the Department of IT, BT and ST, Government of Karnataka, Ref no: 3451-00-090-2-22.

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The emerging field of RNA modifications will answer many questions pertaining to the finetuning of gene expression at post-transcriptional level. Recent investigations have revealed the enzymes involved in the process of laying down these RNA marks and the consensus sequence on which they act [7, 19, 21–25]. It is evident that the consensus sequence is far from being sufficient, as only 10% of the sites in the genome bear the m6A mark, suggesting the roles of additional factor(s) that remain to be discovered. Moreover, the time- and tissuedependent regulation of m6A modification needs to be understood. The molecular mechanism(s) acting downstream of these modifications have been elucidated recently with the discovery of reader proteins [26]. Conclusive evidence has emerged that link m6A RNA methylation with splicing and decreased stability of the RNA transcripts, mediated by the ‘m6A-reader’ [26]. A second class of proteins may exist, which specifically binds RNA once m6A is removed; their binding to RNA may get affected owing to the secondary structure changes on RNA owing to m6A addition [65]. The role of m6A thus needs to be defined at various phenotypic levels: whole organism/tissue level, pathway level, machinery level and an elementary molecular level [65]. Although evidence links the m6A mark in development of brain and gametes, as well as metabolic regulation [24], no studies have yet linked m6A RNA modification with diseases like cancer. The precise form of regulation and its synergistic role with other forms of epigenetic modifications, such as DNA and histone methylation, in diseases like cancer, if any, needs to be understood. As the techniques to investigate these modifications become refined, we can expect to see an increasing number of investigations into this interesting world of posttranscriptional RNA regulation.

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