m 6 A RNA methylation: from mechanisms to therapeutic potential

Review20 January 2021free access m6A RNA methylation: from mechanisms to therapeutic potential P Cody He P Cody He orcid.org/0000-0002-6171-810X Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Committee on Immunology, The University of Chicago, Chicago, IL, USA Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Chuan He Corresponding Author Chuan He [email protected] orcid.org/0000-0003-4319-7424 Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Committee on Immunology, The University of Chicago, Chicago, IL, USA Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author P Cody He P Cody He orcid.org/0000-0002-6171-810X Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Committee on Immunology, The University of Chicago, Chicago, IL, USA Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Chuan He Corresponding Author Chuan He [email protected] orcid.org/0000-0003-4319-7424 Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA Committee on Immunology, The University of Chicago, Chicago, IL, USA Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA Search for more papers by this author Author Information P Cody He1,2,3 and Chuan He *,1,2,3 1Department of Chemistry, Department of Biochemistry and Molecular Biology, Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA 2Committee on Immunology, The University of Chicago, Chicago, IL, USA 3Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA *Corresponding author. Tel: +1 773 7025061; E-mail: [email protected] The EMBO Journal (2021)40:e105977https://doi.org/10.15252/embj.2020105977 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract RNA carries a diverse array of chemical modifications that play important roles in the regulation of gene expression. N6-methyladenosine (m6A), installed onto mRNA by the METTL3/METTL14 methyltransferase complex, is the most prevalent mRNA modification. m6A methylation regulates gene expression by influencing numerous aspects of mRNA metabolism, including pre-mRNA processing, nuclear export, decay, and translation. The importance of m6A methylation as a mode of post-transcriptional gene expression regulation is evident in the crucial roles m6A-mediated gene regulation plays in numerous physiological and pathophysiological processes. Here, we review current knowledge on the mechanisms by which m6A exerts its functions and discuss recent advances that underscore the multifaceted role of m6A in the regulation of gene expression. We highlight advances in our understanding of the regulation of m6A deposition on mRNA and its context-dependent effects on mRNA decay and translation, the role of m6A methylation of non-coding chromosomal-associated RNA species in regulating transcription, and the activities of the RNA demethylase FTO on diverse substrates. We also discuss emerging evidence for the therapeutic potential of targeting m6A regulators in disease. Introduction m6A is a chemical derivative of adenosine in RNA that plays important, wide-ranging roles in the regulation of gene expression. m6A modification of RNA occurs in most eukaryotes and is the most prevalent internal mRNA modification in mammals, where it is present at tens of thousands of sites across the transcriptome, at a frequency of 0.15–0.6% of all adenosines (Table 1) (Dominissini et al, 2012; Liu et al, 2014; Ke et al, 2015). The presence of m6A in mRNA was first identified in the 1970s, and the m6A mRNA methyltransferase complex was purified in the 1990s, with METTL3 being identified as a key component (Desrosiers et al, 1974; Lavi & Shatkin, 1975; Perry et al, 1975; Wei et al, 1975; Wei & Moss, 1977; Bokar et al, 1994, 1997). Later work demonstrated that METTL3 forms a heterodimer with METTL14 to convert A to m6A on mRNA (Liu et al, 2014; Ping et al, 2014; Wang et al, 2014b, 2016a, 2016b; Śledź & Jinek, 2016). METTL3 is the catalytically active methyltransferase, with METTL14 playing an essential structural role to facilitate catalysis. The larger methyltransferase holocomplex contains accessory units including WTAP, VIRMA, RBM15A, RBM15B, ZC3H13, and HAKAI, and is predicted to approach 1,000 kDa in size (Patil et al, 2016; Růžička et al, 2017; Guo et al, 2018; ; Wen et al, 2018; Yue et al, 2018). Orthologs of METTL3 and METTL14 have been identified in the sequenced genomes of many animals and plants, as well as some fungi (Bujnicki et al, 2002), and m6A is present in the mRNA of all human and mouse tissues studied so far (Xiao et al, 2019; Liu et al, 2020b). It should be noted that while the vast majority of m6A on mRNA is deposited by METTL3/METTL14, the U6 snRNA m6A methyltransferase METTL16 has also been reported to catalyze the addition of m6A on the structured 3′UTR of MAT2A transcripts (Pendleton et al, 2017; Doxtader et al, 2018; Mendel et al, 2018; Ruszkowska et al, 2018). The METTL3/METTL14 methyltransferase complex typically exhibits a nuclear localization and co-transcriptionally deposits m6A methylation onto mRNA transcripts, as well as other RNA polymerase II (Pol II)-transcribed RNAs, including long non-coding RNAs (lncRNAs) and primary microRNAs (pri-miRNAs) (Alarcón et al, 2015b; Ke et al, 2017; Slobodin et al, 2017; Knuckles et al, 2018). The methyltransferase deposits m6A within on a specific subset of cellular transcripts, primarily near stop codons/start of terminal exons and within unusually long internal exons (Dominissini et al, 2012; Meyer et al, 2012; Ke et al, 2015). Table 1. Statistics for m6A in mRNA. Quantity Value References Notes Ratio of m6A to A in polyadenylated RNA 0.15–0.6% (LC-MS/MS) Liu et al (2014, 2018, 2020b), Wang et al (2014b), Lin et al (2017), Du et al (2018), Wei et al (2018) m6A to A ratios in polyadenylated RNA are heavily influenced by the highly expressed genes in the transcriptome. In humans, a small fraction of all expressed genes (10–1,000 genes, depending on the tissue type) make up the majority of the mRNA pool (Ramsköld et al, 2009) Percentage of transcripts in the transcriptome that contain m6A ~ 25 to ~ 60% (m6A-seq) Dominissini et al (2012, unpublished) Dominissini et al identified m6A in transcripts of ~ 7,000 genes. The 25% figure is derived from dividing 7,000 from the estimated number of total human genes, ~ 20,000–25,000. However, this is likely an underestimate. First, all genes are not expressed in any given cell type. Second, m6A in relatively lowly expressed genes escapes detection due to low sequence coverage (increasing sequencing depth increases the number of m6A peaks identified). By restricting the analysis to a set of transcripts above an expression cutoff set to the lower quartile of expression for detected methylated transcripts, the percentage of transcripts containing at least one m6A peak is higher, ~ 60% (unpublished). It is important to note here that this percentage reflects the number of genes whose transcripts exhibit m6A, not the stoichiometry of m6A in the transcripts of any given gene Average number of m6A per transcript ~ 3 m6A residues per average mRNA transcript (LC-MS/MS), ~ 1.7 m6A-seq peaks per transcript (m6A-seq) Perry et al (1975), Dominissini et al (2012) It is important to consider that these are averages. Many transcripts are unmethylated and many have significantly more m6A peaks than the average Percentage of DRACH motifs that are methylated ~ 5% Dominissini et al (2012) Dominissini et al estimate that a prevalence of ~ 1 m6A peak every 2,000 bp. The number of DRACH motifs found in 2,000 bp of sequence is theoretically (3/4)(2/4)(1/4)(1/4)(2/4)*2,000 = 23.4. Thus, the percentage is estimated at 1/23.4 ~ 5%. The actual percentage may be several-fold higher or lower, given that m6A peaks may contain several m6A sites and the true frequency of DRACH motifs may diverge from the theoretical estimate The effects of m6A on gene expression are wide-ranging (Fig 1). m6A alters pre-mRNA processing, promotes mRNA nuclear export, alters mRNA stability, increases translation efficiency, and facilitates non-canonical translation initiation (Wang et al, 2014a, 2015; Liu et al, 2015, 2017; Meyer et al, 2015; Xiao et al, 2016; Barbieri et al, 2017; Roundtree et al, 2017; Slobodin et al, 2017; Kasowitz et al, 2018; Zhou et al, 2018, 2019). Some of these effects are mediated by m6A “reader” proteins that can selectively recognize m6A and exert a regulatory function on the m6A-marked mRNA. m6A readers include members of the YTH family, which bind m6A with their eponymous YTH domains (Wang et al, 2014a, 2015; Xiao et al, 2016; Hsu et al, 2017; Roundtree et al, 2017; Shi et al, 2017). Deposition of m6A on RNA can also alter local RNA secondary structure. Here m6A can exert stabilizing and destabilizing effects, depending on its sequence context, but results so far indicate that destabilizing effects are more predominant transcriptome-wide (Kierzek & Kierzek, 2003; Roost et al, 2015; Spitale et al, 2015; Sun et al, 2019b). The RNA-binding proteins (RBPs) HNRNPC and HNRNPG are indirect “structural-switch” readers, that can selectively bind at “structural switches”, sites at which m6A alters the accessibility of proximal RNA sequence to facilitate binding of RBPs (Liu et al, 2015, 2017; Zhou et al, 2019). Several other RBPs that preferentially bind m6A have also been identified, but beyond the YTH family and HNRNPC/G, how these proteins selectively bind m6A has not been conclusively elucidated (Alarcón et al, 2015a; Ae et al, 2017; Edupuganti et al, 2017; Wu et al, 2018a). More recently, it has furthermore been suggested that m6A may also regulate gene expression by disfavoring the binding of certain RBPs to mRNA, as has been reported for G3BP1 and LIN28A (Edupuganti et al, 2017; Sun et al, 2019b). The wide array of mRNA processing factors whose activities are regulated by m6A enable m6A to regulate the large number of diverse molecular processes involved in gene expression. In addition to m6A-mediated regulation of gene expression at the mRNA level through the direct recruitment of reader proteins to the transcript, it has also been recently discovered that m6A modification of non-coding chromosome-associated RNA transcripts functions to modulate gene expression by regulating transcription of nearby mRNAs (Liu et al, 2020a). Thus, it is becoming increasingly clear that m6A has numerous effects on gene expression through diverse mechanisms. Figure 1. m6A is a multifaceted regulator of gene expression m6A (red circle) regulates transcription, alternative splicing, alternative polyadenylation, nuclear export, cap-dependent and cap-independent translation, mRNA degradation, and mRNA stabilization. A diverse set of reader proteins that selectively bind m6A, either directly or indirectly, mediate these multifaceted effects on gene expression. Note that for clarity, nuclear processes are shown to occur after release of RNA from the polymerase, but some of the depicted nuclear processes may also occur co-transcriptionally. Download figure Download PowerPoint An important facet of m6A methylation as a regulatory system is its reversibility. m6A on mRNA can be removed by FTO and ALKBH5, m6A demethylases, posing an additional dimension of regulation for the m6A epitranscriptome (Jia et al, 2011; Zheng et al, 2013). Both are iron- and α-ketoglutarate-dependent dioxygenases that mediate oxidative demethylation of m6A. FTO has been demonstrated to demethylate m6A on mRNA, m6Am on mRNA and snRNAs, and m1A on tRNA (Mauer et al, 2017, 2019; Wei et al, 2018). Due to its wide array of substrates, the activities of FTO that are most relevant for its crucial in vivo functions have been the subject of debate, as discussed later in this review. Given its wide-ranging roles in the regulation of gene expression, it is unsurprising that m6A is required for numerous physiological and pathophysiological processes. Depletion of METTL3 homologs leads to defective meiosis in yeast, and developmental arrest in flies and plants (Zhong et al, 2008; Hongay & Orr-Weaver, 2011; Agarwala et al, 2012; Bodi et al, 2012; Schwartz et al, 2014). Genetic knockout of either Mettl3 or Mettl14 is developmentally lethal in mice, with embryos failing to thrive at around E5.5 (Batista et al, 2014; Geula et al, 2015). Analysis of tissue-specific knockout mouse models of Mettl3 and Mettl14 has revealed essential roles for m6A in brain development and function, cardiac homeostasis, immune system development and function, spermatogenesis, and skeletal function (Lin et al, 2017; Yoon et al, 2017; Li et al, 2017a; Rubio et al, 2018; Wang et al, 2018a, 2018c, 2019; Wu et al, 2018b; Dorn et al, 2019; Winkler et al, 2019; Xu et al, 2020). Moreover, knockout of either Mettl3 or Mettl14 severely blocks or delays differentiation in numerous stem cell or progenitor cell systems, including embryonic stem cells, embryonic neuronal stem cells (Yoon et al, 2017; Wang et al, 2018c), hematopoietic stem cells (Vu et al, 2017; Zhang et al, 2017a; Weng et al, 2018a; Cheng et al, 2019; Lee et al, 2019), naïve T cells (Li et al, 2017a), and bone marrow mesenchymal stem cells (Wu et al, 2018b). Additionally, m6A has been implicated in the pathogenesis of a variety of diseases, including in numerous cancer types and in type 2 diabetes (Batista, 2017; De Jesus et al, 2019; Ianniello et al, 2019; Yang et al, 2019b; Huang et al, 2020). Three independent reports implicate METTL3 or METTL14 as important regulators of AML, suggesting that targeting m6A may hold therapeutic potential in this setting (Barbieri et al, 2017; Vu et al, 2017; Weng et al, 2018a). Overall, m6A has emerged as a key regulator of numerous important biological processes in normal physiology and in disease. In this review, we will delve into recent evidence that underscores the multifaceted nature of the role of m6A in regulating gene expression. We highlight new advances in our understanding of m6A deposition on mRNA and its effects on mRNA decay and translation, the role of m6A on non-coding chromosomal-associated RNA species in regulating mRNA transcription, the activities of the RNA demethylase FTO on diverse substrates, and discuss the therapeutic potential of targeting m6A regulators. Regulation of m6A deposition on mRNA As mentioned above, a notable characteristic of the METTL3/METTL14 methyltransferase is the specificity with which it deposits m6A on the transcriptome. The consensus motif for METTL3/METTL14 is a commonly occurring DRACH (D = A, G or T; R = A or G; H = A, C or U) consensus sequence, but only a fraction of sequences that contain this consensus motif are methylated (Table 1). Further, m6A exhibits a marked regional bias in its distribution across the transcriptome. m6A can be found throughout the length of transcripts, but is strongly enriched in the vicinity of stop codons and in unusually long internal exons (Fig 2A) (Ke et al, 2017). According to one analysis, internal exons that are > 200 nt comprise only ~ 15% of all internal exons, but internal exons > 200 nt contain ~ 80% of all m6A sites present within internal exons (Ke et al, 2017). This cannot be explained by overrepresentation of methyltransferase motifs in long internal exons, because internal exons > 200 nt only contain ~ 30% of all motifs. Additionally, transcript isoforms that use proximal alternative polyadenylation sites are generally enriched for m6A over longer isoforms using distal alternative polyadenylation sites (Molinie et al, 2016). These observations imply regulation of m6A deposition by METTL3/METTL14 that extends beyond primary sequence constraints. Figure 2. Specificity of the m6A epitranscriptome (A) Schematic representing the distribution of m6A in the mammalian transcriptome. A subset of transcripts contain one or more m6A sites, while another subset are not methylated. m6A is enriched in unusually long internal exons and near stop codons/start of last exons. (B) Deposition is regulated by intrinsic factors, such as the preference of the METTL3/METTL14 methyltransferase for specific RNA sequences. m6A deposition is also regulated by external factors; transcription factors, RNA-binding proteins, RNA polymerase II, and the H3K36me3 histone modification have been reported to recruit the METTL3/METTL14 methyltransferase to mRNAs to promote methylation. m6A demethylases FTO and ALKBH5 can also tune m6A levels at a subset of sites following their initial deposition by METTL3/METTL14. Download figure Download PowerPoint Determinants of m6A specificity are central to m6A-mediated regulation of gene expression, as the restriction of m6A to a subset of cellular transcripts (estimated at 25–60%) allows for selective regulation of specific transcripts by m6A reader proteins (Table 1). For example, in mouse embryonic stem cells (mESCs), 80% of genes that regulate naïve pluripotency exhibit m6A methylation on their transcripts. This is thought to facilitate their clearance during the termination of pluripotency to enable embryonic stem cell differentiation (Geula et al, 2015). Further, the specific position at which an mRNA is m6A methylated may also be relevant for gene expression, since the nature of m6A-mediated regulation appears to differ depending on the location of m6A within the transcript (Wang et al, 2014a, 2015; Meyer et al, 2015; Choi et al, 2016; Barbieri et al, 2017; Slobodin et al, 2017; Zhou et al, 2018; Mao et al, 2019). Given the relevance of m6A specificity for gene expression regulation, several recent studies have focused on elucidating the molecular mechanisms that govern regulation of m6A deposition in various cellular contexts. Certain transcription factors can promote methylation of specific transcripts by recruiting the methyltransferase complex to their target loci (Barbieri et al, 2017; Bertero et al, 2018). In addition, RBPs, such as TARBP2, can recruit the methyltransferase complex to its bound transcripts (Fish et al, 2019) and slow transcription rates have been proposed to promote m6A deposition by enhancing co-transcriptional recruitment of the methyltransferase complex to its RNA substrate (Slobodin et al, 2017). The histone H3K36me3 modification has also been proposed to recruit the methyltransferase complex to chromatin in order to promote methylation of nascent RNAs (Huang et al, 2019a). Collectively, these papers have shed light on how diverse biological inputs can alter m6A deposition and lead to differential downstream effects on gene expression and cellular phenotypes (Fig 2B). The wide variety of mechanisms that affect m6A methylation support the notion that m6A deposition is a highly regulated event that is interwoven with diverse cellular processes. While progress has been made in elucidating new mechanisms regulating m6A deposition, fundamental questions regarding m6A deposition remain unanswered. Notably, the general rules that govern m6A deposition and generate the observed distribution of m6A on the transcriptome are still poorly understood. First, the degree to which intrinsic vs. extrinsic factors determine m6A levels has been the subject of debate: to what extent are m6A levels determined by the intrinsic preference of the METTL3/METTL14 methyltransferase for certain nucleotide sequences vs. the contribution of extrinsic regulation of methyltransferase complex activity by external factors such as RBPs, transcription factors, RNA polymerase, or other yet to be discovered factors? This question is important due to its potential implications for m6A as a regulatory system. If intrinsic determinants are dominant, this would suggest that m6A deposition is not highly regulated, and would generally operate independently of other cellular processes. It would suggest that global changes in m6A methylation could occur if enzyme levels or other reaction variables change, but relative changes of m6A levels at different sites would be limited. Conversely, if extrinsic determinants dominate, this would indicate a more dynamic role for m6A, in which m6A levels may react to changes in the activity of external factors in different cellular states. It would moreover imply that m6A deposition is interwoven with other cellular processes, allowing for subsets of sites to gain or lose methylation in response to specific cellular events. The involvement of extrinsic determinants is supported by a large body of literature that reports differential methylation of significant numbers of m6A sites in different cellular contexts (Xiao et al, 2019; Liu et al, 2020b). Additionally, several mechanisms through which trans-acting factors can regulate methylation have been characterized, as described above. However, other reports have concluded that intrinsic determinants are predominant and that levels may be largely “hard-coded” in cis by the local sequence surrounding the m6A site (Schwartz et al, 2014; Garcia-Campos et al, 2019). Part of this apparent discrepancy may be due to differences in m6A regulation in different organisms. Most studies characterizing the importance of extrinsic factors have been carried out in mammalian cells (Barbieri et al, 2017; Slobodin et al, 2017; Bertero et al, 2018; Fish et al, 2019). One study reporting that intrinsic determinants largely control m6A levels primarily examined the ability of local sequence features to predict m6A methylation levels in the budding yeast Saccharomyces cerevisiae (Garcia-Campos et al, 2019). The authors found that in mammalian systems, the ability to predict m6A methylation levels from local sequence features was diminished compared to the ability to predict methylation levels in S. cerevisiae. Part of this discrepancy may also arise from an assumption that local sequence variation only alters intrinsic determinants and not regulation by extrinsic factors, which may not necessarily hold true. Overall, we propose that intrinsic determinants and extrinsic determinants likely both play significant roles in shaping the landscape of m6A on the transcriptome and that their relative contributions likely vary based on the specific site and cellular context. Future work using systematic approaches to directly assess the contributions of intrinsic and extrinsic determinants of m6A will advance our understanding of the overall determinants and general characteristics of m6A regulation. Since batch effects and other variables can cause significant variation in m6A measurements, ensuring the use of optimal experimental designs and appropriate statistical models for differential methylation analysis may help bring more clarity to this issue (Zhang et al, 2019). Further development and application of robust and quantitative high-throughput methods of m6A detection will also enable greater understanding of m6A regulation by enhancing the capability to detect changes in methylation stoichiometry under different conditions. We should note that beyond regulation of m6A deposition itself, an additional layer of regulation may be mediated by the differential activities of diverse m6A reader proteins. While extrinsic determinants may facilitate m6A dynamics in response to cell differentiation and signals from stimulation or stress, m6A deposited via both intrinsic and extrinsic determinants could be recognized by readers differently under different cellular contexts. Thus, even if m6A levels at a particular site do not change, the functional effect of that m6A site may still vary according to the cell state. For example, m6A readers could compete for binding to m6A sites, in which case differing levels or ratios of readers in different cell types could lead to diverging gene expression outcomes. In this context, post-translational modifications of readers also may tune their cellular localization, m6A binding affinity and interacting protein partners. All these variables could affect the functional outcome of mRNA m6A regulation (Fig 3). Figure 3. Theoretical model for how changes in cellular state may impact m6A regulation m6A regulatory factors can act at multiple levels to enable differential gene expression regulation. Upon a change in cell state, m6A sites that are extensively regulated by extrinsic determinants (e.g., RNA-binding proteins that recruit METTL3/METTL14 to the site) may vary in their methylation level if the levels or activities of these factors change. In contrast, m6A sites that are primarily controlled by intrinsic determinants may exhibit stable methylation. Additionally, levels or activities of m6A readers may vary upon a change in cell state. This may result in differential reader binding to m6A sites. Given the diverse activities of various m6A readers, the effect of a particular m6A site on mRNA metabolism may change even if methylation levels remain stable. Download figure Download PowerPoint Another major unknown in the regulation of m6A deposition is the mechanism(s) that govern the specific enrichment of m6A near certain transcriptomic features. Despite recognition of the strong enrichment of m6A within unusually long exons and near stop codons since the first transcriptome-wide m6A mapping studies, the mechanisms regulating m6A methylation reported to date do not explain how m6A is selectively deposited in this specific distribution. The m6A methyltransferase accessory factor VIRMA appears to promote methylation of many sites in mRNA 3′ untranslated regions (UTRs) and near stop codons, but the mechanistic basis for this effect is to date still unclear (Yue et al, 2018). H3K36me3 directly recruits METTL14 to gene bodies to promote m6A deposition, and it does not appear to confer enrichment of m6A near stop codons, as this enrichment is still observed when H3K36me3 levels are reduced (Huang et al, 2019a). The inability of currently known mechanisms to explain how m6A is specifically enriched in certain transcriptome regions suggests that major mechanisms regulating m6A deposition globally remain to be discovered. Most reported mechanisms of m6A deposition regulation involve models in which a trans-acting factor recruits the METTL3/METTL14 methyltransferase to RNAs in order to promote methylation at proximal sites. However, mechanisms with different modes of action may need to be considered in order to explain m6A specificity at distinct transcriptomic features. Future work on the mechanisms that govern m6A specificity will advance the current understanding of m6A-mediated gene regulation. As METTL3/METTL14 is the predominant m6A methyltransferase on mRNA, we have mainly focused on METTL3/METTL14-catalyzed m6A in this review. However, it should be noted that other methyltransferases that methylate the N6 position of adenosine on various RNA species have recently been discovered and characterized. PCIF1 (phosphorylated CTD interacting factor 1), an RNA polymerase II-associated factor that contains a N6-methyladenosine methyltransferase domain, mediates the methylation of N6, 2′-O-dimethyladenosine (m6Am), which occurs near the mRNA cap (Akichika et al, 2019; Boulias et al, 2019; Sendinc et al, 2019; Sun et al, 2019a). 5′ ends of eukaryotic mRNAs carry a 7-methylguanosine (m7G) cap linked to the rest of the mRNA by a triphosphate linkage and it is well known that the first nucleotide after the m7G cap can be methylated on the ribose sugar. If that nucleotide is adenosine, it can be further methylated to m6Am by PCIF1. Additional characterized m6A methyltransferases include METTL16, ZCCHC4, METTL5, and METTL4. METTL16 deposits m6A methylation on U6 snRNA and on MAT2A mRNA (Pendleton et al, 2017; Doxtader et al, 2018; Mendel et al, 2018), ZCCHC4 and METTL5 are rRNA m6A methyltransferases (Ma et al, 2019; van Tran et al, 2019; Ignatova et al, 2020; Leismann et al, 2020), and METTL4 mediates internal m6A and m6Am methylation of U2 snRNA (Chen et al, 2020; preprint: Goh et al, 2020). Context-dependent roles of m6A in mRNA decay and translation m6A is known to regulate mRNA decay and translation through a variety of mechanisms. The mammalian YTHDF family of proteins, consisting of YTHDF1, YTHDF2, and YTHDF3, are cytosolic m6A readers that regulate m6A degradation and translation. YTHDF2, the first to have been characterized, selectively recognizes and shuttles m6A-modified RNAs for degradation by multiple mechanisms (Wang et al, 2014a). YTHDF2 recruits the CCR4-NOT deadenylase complex to promote deadenylation and degradation of m6A-marked mRNAs and also recruits the RNase P/MRP complex to promote endoribonucleolytic cleavage of m6A-marked mRNAs (Du et al, 2016; Park et al, 2019). Whereas YTHDF2 mediates mRNA decay, the two other cytosolic YTHDF proteins, YTHDF1 and YTHDF3, facilitate translation of their target methylated mRNAs. YTHDF1 promotes translation by recruiting the translation initiation factor eIF3 to bound mRNAs (Wang et al, 2015). YTHDF3 in turn interacts with YTHDF1 and is thought to shuttle its bound transcripts to YTHDF1 to promote their translation (Shi et al, 2017). In addition to this YTHDF1/3-mediated regulation, other reported mechanisms of m6A-mediated translation regulation include the direct binding