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The Epitranscriptome in Regulation

Eyal Peer, Sharon Moshitch-Moshkovitz, Gideon Rechavi, and Dan Dominissini

Sackler School of Medicine, Tel Aviv University, Tel Aviv 6997801, Israel; Research Center and Wohl Centre for Translational Medicine, Chaim Sheba Medical Center, Tel-Hashomer 5262160, Israel Correspondence: [email protected]

The cellular proteome reflects the total outcome of many regulatory mechanisms that affect the metabolism of messenger RNA (mRNA) along its pathway from synthesis to degradation. Accumulating evidence in recent years has uncovered the roles of a growing number of mRNA modifications in every step along this pathway, shaping translational output. mRNA modifications affect the translation machinery directly, by influencing translation initiation, elongation and termination, or by altering mRNA levels and subcellular localization. Features of modification-related translational control are described, charting a new and complex layer of translational regulation.

ellular homeostasis requires regulation of introduced the novel notion that internal chem- Cprotein levels, as impaired regulation can ical modifications of mRNA and long noncod- result in cellular death or dysregulated prolifer- ing RNA (lncRNA) are potentially dynamic and ation (Hershey et al. 2012). Protein levels are a sometimes reversible events, which constitute consequence of three fundamental factors: the essential regulatory elements in basic RNA-pro- abundance of messenger RNA (mRNA), the cessing steps such as splicing, transport, trans- efficiency of translation, and protein stability. lation, and decay. The epitranscriptome, as this First isolated from ribosomal RNA (rRNA) ensemble is now known, comprises a growing almost seven decades ago (Cohn 1951), modi- number of chemical adducts: m6A, inosine (I), fied RNA bases were later also discovered to 5-methylcytidine (5mC), 5-hydroxymethylcyti- be abundant constituents of mRNA, with N6- dine (5hmC), (Ψ), 20-O-methyl- methyladenosine (m6A) as the most prevalent ation (Nm), and N1-methyladenosine (m1A). internal mRNA modification (Desrosiers et al. This review focuses on m6A as well as on more 1974). To date, 170 different modified nucleo- recently characterized mRNA modifications and tides have been identified in RNA from all types their effects on translation. and species (Boccaletto et al. 2018), expanding the RNA alphabet to embed transcripts with N6-METHYLADENOSINE (m6A) additional information. The introduction of -wide mapping methods and the m6A is the most common internal (noncap) discovery of capable of removing m6A mRNA modification found in eukaryotic or- and of dedicated m6A-binding proteins have ganisms as well as in RNA of nuclear-replicat-

Editors: Michael B. Mathews, Nahum Sonenberg, and John W.B. Hershey Additional Perspectives on Translation Mechanisms and Control available at www.cshperspectives.org Copyright © 2019 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a032623 Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032623

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ing (reviewed in Fu et al. 2014). m6A ing family protein 1 (YTHDF1) and YTH do- constitutes 0.1%–0.5% of residues in main-containing family protein 3 (YTHDF3) mRNA, corresponding to ∼3–5m6A modifica- (regulation of translation); YTH domain-con- tions per transcript. Although m6A was discov- taining family protein 2 (YTHDF2) (regulation ered in mRNA decades ago (Desrosiers et al. of RNA turnover); and heterogeneous nuclear 1974), its study was jump-started in recent years ribonucleoprotein A2/B1 (HNRNPA2B1) and with the identification of the m6A-specific de- YTH domain-containing 1 (YTHDC1) (pro- methylases: the fat mass and obesity-associated cessing of primary and alternative protein (FTO) and AlkB homolog 5 (ALKBH5) splicing) (Meyerand Jaffrey 2017). These discov- (Jia et al. 2011; Zheng et al. 2013), followed by eries opened up the field of epitranscriptomics the development of high-throughput m6A map- for accelerated investigation. ping tools, m6A-seq (Dominissini et al. 2012) and methylated RNA immunoprecipitation and PSEUDOURIDINE (Ψ) sequencing (MeRIP-Seq) (Meyer et al. 2012). Transcriptome-wide mapping of m6A revealed Ψ was the first posttranscriptional modification an evolutionarily conserved, nonrandom distri- to be identified and, taking all classes of RNA bution (Dominissini et al. 2012; Meyer et al. into account, is also the most abundant. Recent- 2012); m6A preferentially decorates 50 untrans- ly it was shown to be more abundant in mRNA lated regions (UTRs), long internal , and than previously believed, with a Ψ/U ratio the vicinity of stop codons. Sites preferentially of 0.2%–0.6% in mammalian mRNA (Li et al. appear within the consensus sequence RRACU 2015). It is formed by isomerization of in (R = A or G) (Dominissini et al. 2012). which the base is rotated 180° along the N3-C6 The deposition of m6A in mRNA is per- axis. Although the modified base has unaltered formed by a multicomponent Watson–Crick base-pairing properties, it gains complex of which several components have been an additional hydrogen-bond donor at its non- identified: methyltransferase-like 3 (METTL3), Watson–Crick edge, thus endowing it with the catalytic component; methyltransferase- distinct chemical properties (Ge and Yu 2013). like 14 (METTL14), an RNA adaptor needed Ψ plays roles in the biogenesis and function for METTL3 activity; Wilms Tumor 1–associat- of spliceosomal small nuclear (snRNAs) ing protein (WTAP), a regulatory factor that and rRNA. Methods for transcriptome-wide is also responsible for nuclear speckle localiza- mapping of Ψ using Ψ-specific chemical labeling tion; RNA-binding motif (RBM) proteins 15/15B by CMC [N-cyclohexyl-N0-(2-morpholinoethyl) (RBM15/15B), mediators of specif- carbodiimide metho-p-toluenesulfonate] iden- icity by directing the complex to specific tran- tified hundreds of Ψ sites in yeast and human scripts; and KIAA1429/Virilizer, whose func- mRNAs (Carlile et al. 2014; Schwartz et al. 2014; tion is still unclear (reviewed in Meyer and Li et al. 2015). No preferred localization of Ψ Jaffrey 2017). to specific regions of mRNA was shown. The balance between writing (adding) and Installation of Ψ is catalyzed by pseudo- erasing m6A from transcripts confers upon uridine synthases (PUSs) and can be achieved this modification a dynamic nature, which is ev- through two distinct mechanisms (Li et al. 2016a): ident under stress conditions (Dominissini et al. (1) RNA-independent pseudouridylation that is 2012), supporting a role in regulation of gene catalyzed by a single PUS responsible expression. Indeed, m6A plays a role in many for both substrate recognition and catalysis; or aspects of , affecting RNA splic- (2) RNA-dependent pseudouridylation that re- ing, nuclear export and retention, translation, lies on RNA–protein complexes consisting of and turnover. The major mechanism by which box H/ACA noncoding RNAs in conjunction m6A exerts its effects is by recruiting m6A reader with the centromere-binding factor 5 (Cbf5)/ proteins: fragile X mental retardation 1 (FMR1) Dyskerin protein complex. Although mRNA Ψ (Edupuganti et al. 2017); YTH domain-contain- is dynamically regulated in response to environ-

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The Epitranscriptome in Translation Regulation

mental signals (Carlile et al. 2014), pseudouri- to a GUUCRA tRNA-like motif (R = A or G) dylation has not been shown to be reversible. and have T-loop-like structures, are evenly dis- tributed along transcript segments, and consti- fi N1-METHYLADENOSINE (m1A) tute roughly 10% of all identi ed sites. Last, TRMT61B-dependent m1A sites are primarily Using mass spectrometry and a mapping meth- located within the coding region of mitochon- odology based on immunocapture and massively drial mRNA, where they inhibit translation parallel sequencing, m1A was recently identified (Li et al. 2017b). as a new epitranscriptome marker that occurs on thousands of human and mouse mRNAs 20-O-METHYLATION (Nm) (Dominissini et al. 2016; Li et al. 2016b). m1A was known to exist in transfer RNA (tRNA) The ribose ring can be methylated at the 20 po- and rRNA and is unique in that it has both sition to form Nm (Boccaletto et al. 2018), a methyl group and a positive charge under an abundant modification present in mRNA, physiological conditions, which could markedly rRNA, tRNA, snRNA, and microRNA, which alter RNA structure and protein–RNA interac- is essential for their biogenesis, metabolism, tions. Transcriptome-wide mapping of m1A and function (Liang et al. 2009; Daffis et al. revealed unique features of its distribution that 2010; Lin et al. 2011; Züst et al. 2011; Deryu- strongly indicate functional roles: (1) m1Ais sheva et al. 2012; Jöckel et al. 2012; Somme et al. strongly enriched in mRNA 50UTRs, close to 2014). Nm modification is catalyzed either by translation initiation sites; (2) m1A is present in stand-alone that recognize highly structured regions of 50UTRs; (3) m1A their targets according to sequence and struc- positively correlates with translation efficiency ture (Somme et al. 2014), or by the enzyme and protein levels; (4) the distribution of m1Ais fibrillarin, part of a ribonucleoprotein complex highly conserved in all mouse and human that is guided to its targets by different C/D-box types examined; and (5) m1A is dynamic in re- small nucleolar RNAs (snoRNAs) (Shubina sponse to stress and physiological signals, and et al. 2016). Nm also occurs at the 50 cap its level varies across tissues (Dominissini (Byszewska et al. 2014) and internal positions et al. 2016; Li et al. 2016b). Together, these at- of non-rRNA transcripts (Lacoux et al. 2012). tributes suggest a positive and dynamic role for 20-O-methylation endows nucleotides with m1A in translation initiation in mammalian greater hydrophobicity, protects against nucleo- cells. lytic attack, and stabilizes RNA helices (Kumar A recently improved detection method iden- et al. 2014; Yildirim et al. 2014). tified hundreds of m1A sites with single-nucle- In higher , the 50 penultimate otide resolution; most of them are in the mRNA (the first transcribed) and antepenultimate 50UTR (including a minor fraction in the first (the second transcribed) nucleotides in mRNA transcribed nucleotide, cap + 1), validating and (m7GpppNmNm) may be 20-O-methylated by refining earlier studies. Sites fall into three sub- stand-alone enzymes that recognize the cap. sets defined by their location, the identity of Aside from these Nm sites, accumulating evi- the writer enzyme, and sequence-structure fea- dence suggests that internal positions in mRNA tures: TRMT6/61A-independent and -dependent can also be 20-O-methylated (Gumienny et al. m1A sites in nuclear-encoded mRNA; and 2016). This possibility is especially intriguing in tRNA methyltransferase 61B (TRMT61B)-de- light of the consequences that such modification pendent m1A sites in mitochondrial-encoded could have when present in the coding sequence mRNA. tRNA methyltransferase 6 (TRMT6)/ (CDS) (Hoernes et al. 2016). tRNA methyltransferase 61A (TRMT61A)-in- A recently developed Nm mapping method, dependent m1A sites constitute the largest sub- Nm-Seq, uncovered thousands of Nm sites in set and are strongly enriched in the 50UTR. human mRNA, with Um being the dominant TRMT6/61A-dependent m1A sites conform species (Dai et al. 2017). Nm sites are enriched

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in the 30UTR and around internal splice sites. In sponsible for 5mC in some mRNAs as well the CDS, the sites are nonrandomly distributed (Hussain et al. 2013; Khoddami and Cairns with 60.4% of them occurring in only six 2013). codons, suggesting functional roles. Recently, ALYREF, a nuclear export factor, has been identified as a reader of 5mC in mRNA N6,20-O-DIMETHYLADENOSINE (m6Am) (Yang et al. 2017a). Although it is not known to be completely reversible, it was discovered The 50 end of mRNA transcripts is modified by that 5mC in RNA undergoes oxidation by the an N7-methylguanosine (m7G) cap and 20-O ten-eleven translocation (Tet) protein family methylation (Nm) of the ribose sugar of the first, (TET1,2,3) to produce 5hmC (Fu et al. 2014). and sometimes the second, transcribed nucleo- Transcriptome-wide mapping of 5hmC in tides (Keith et al. 1978) as mentioned above. Drosophila melanogaster was performed by Cap-associated modifications recruit translation adapting MeRIP-seq to 5hmC (hMeRIP-seq), initiation factors to mRNA and allow the cell to and yielded over 3000 putative 5hmC sites in discriminate from viral mRNA, 20-O-meth- mRNA. Although the function of 5hmC is un- ylation being especially important for the latter clear, ribosomal profiling (see Ingolia et al. (Daffis et al. 2010). When the first transcribed 2018) revealed that 5hmC is highly enriched in nucleotide (the 50 penultimate base) is Am, actively translated transcripts, suggesting that it can be further methylated at the N6 position 5hmC may facilitate mRNA translation (Delatte by an unidentified methyltransferase to form et al. 2016). m6Am (Keith et al. 1978). Taking advantage fi 6 of the af nity for m Am of an antibody raised READING THE EPITRANSCRIPTOME against m6A, hundreds of m6Am sites in mRNAs have recently been mapped (Lacoux et al. 2012). RNA modifications exert their effects, both di- m6Am in the 50 cap is dynamic and reversible; rectly and indirectly, by altering the chemistry of it can be demethylated to Am by FTO (Mauer RNA nucleotides. A modified RNA nucleotide et al. 2017) to affect transcript decapping and can be specifically recognized by certain RNA- thereby mRNA stability. binding proteins, which then act as readers of the modification and determine the fate of the fi 5-METHYLCYTIDINE (5mC) AND modi ed mRNA. When the effect is mediated 5-HYDROXYMETHYLCYTIDINE (5hmC) by binding proteins, it is termed indirect. It can also stabilize or destabilize the structure of an Methylation of cytosine at the fifth position was RNA molecule, leading to a direct change in identified in mRNA more than 40 years ago level, function, or even translation dynamics. (Dubin and Taylor 1975; Dubin et al. 1977) The effect can also be mediated by a combina- but other studies failed to recapitulate the find- tion of direct and indirect mechanisms, where a ing (Desrosiers et al. 1975; Wei et al. 1976) and change in RNA structure improves the accessi- some have hypothesized that the detection of bility for RNA-binding proteins. In addition, 5mC in mRNA was merely because of contam- hydrophobic modifications incur a solvation ination from other RNA species (Bokar 2005). penalty in water, which can be reduced by However, appropriating bisulfite sequencing—a interaction with protein residues containing method used in mapping 5mC in DNA—for hydrophobic side-chains (Noeske et al. 2015; transcriptome-wide mapping of 5mC in RNA Roundtree et al. 2017). confirmed 5mC as an mRNA modification Each modification carries distinct chemical and unveiled thousands of sites with nonran- implications. In m6A, methylation at the sixth dom distribution (Squires et al. 2012; Amort position of the base does not alter hydrogen- et al. 2017). NOP2/Sun RNA methyltransferase bonding donors and acceptors and does not family member 2 (NSUN2), a methyltransferase appear to affect translational fidelity (You et al. that installs 5mC in tRNA, was identified as re- 2017), but the added methyl group creates steric

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The Epitranscriptome in Translation Regulation

hindrance, thereby changing the energetics of the effect of m6A on mRNA stability (Wang the AU pair and destabilizing the pairing with et al. 2014; Du et al. 2016; Shi et al. 2017), uracil (Roost et al. 2015). In m1A, methylation translation (Wang et al. 2015; Li et al. 2017a; at the N1 position results in a positive charge, Shi et al. 2017), and processing (Xiao et al. potentially leading to strong electrostatic inter- 2016), as well as non-mRNA-related roles such actions (He et al. 2005). Additionally, and in as lncRNA-mediated transcriptional repression contrast to m6A, the added methyl group pro- (Patil et al. 2016) and translation of circular trudes from the Watson–Crick hydrogen-bond- RNA (circRNA) (Yang et al. 2017b; Chekulaeva ing face of adenine, causing the nucleotide to and Rajewsky 2018). Although only the YTH remain unpaired (Lu et al. 2010). Also in con- domain has been structurally characterized in trast with m6A, m1A appears to have the poten- complex with m6A (Li et al. 2014; Luo and tial to block the translation machinery (You Tong 2014; Theler et al. 2014; Xu et al. 2014; et al. 2017). In Ψ, although the isomerization Zhu et al. 2014), other m6A readers have been does not change the Watson–Crick base-pair- suggested (Edupuganti et al. 2017; Huang et al. ing, it does add another potential hydrogen- 2018). bond donor to the base (Ge and Yu 2013). Methylation of the ribose in Nm stabilizes INDIRECT AND DIRECT EFFECTS OF mRNA ’ RNA helices and augments the nucleotide s hy- MODIFICATIONS ON TRANSLATION drophobicity, protecting it from nucleolytic at- tack (Kumar et al. 2014; Yildirim et al. 2014). The translation rate of a protein is proportional The direct and indirect mechanisms by to the concentration and translational efficiency which modifications affect mRNA metabolism of its mRNA (Hershey et al. 2012). mRNA mod- and translation have been explored mainly for ifications define the proteome by influencing m6A. m6A can restructure RNA to control ac- these two factors. They play a direct role by at- cessibility of sequence motifs to RNA-binding tracting translation initiation factors, influenc- proteins. This dynamic interdependence be- ing translation elongation and termination, and tween RNA structure and modification, termed potentially recoding the , and an the m6A-switch, has functional consequences. indirect role by altering mRNA stability, splic- For example, heterogeneous nuclear ribonu- ing, nuclear export, and subcellular localization. cleoprotein (hnRNP) C does not bind m6A but 6 rather a U-tract opposing the m A site, made Indirect Effects accessible by m6A. Global reduction of m6A levels masks a significant subset of hnRNP C The levels of mRNAs can be modulated by binding sites, thereby affecting hnRNP C’s effect a change in their stability conferred by several on (Liu et al. 2015). mRNA modifications; the half-life of m6A- Genuine direct m6A readers have been methylated transcripts is on average shorter discovered and studied, starting with identifica- than nonmethylated ones. Degradation of these tion of YTH domain family proteins as bona fide transcripts is mediated by YTHDF2 that prefer- m6A readers (Dominissini et al. 2012). The YTH entially binds m6A-containing transcripts and domain is a highly conserved RNA-binding moves them from translatable pools to P-bodies domain, identified in over 170 family members or stress granules (Wang et al. 2014). YTHDF2 in a wide range of eukaryotes, from yeast and recruits the CCR4–NOT deadenylase complex plants to vertebrates, appearing in five human by directly interacting with CNOT1 to initiate proteins: YTHDF1-3 and YTHDC1-2 (Stoilov deadenylation and mRNA degradation (Du et al. 2002; Zhang et al. 2010). Initially charac- et al. 2016; Heck and Wilusz 2018). terized at the turn of the century (Imai et al. In contrast to m6A, the presence of m6Am 1998), YTH was recently validated as an m6A- (Cap1) in mRNA increases the stability of tran- binding domain (Wang et al. 2014) and YTH scripts by conferring resistance to the mRNA domain family proteins were shown to mediate decapping enzyme, DCP2. FTO is able to

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remove the methyl group from the N6 position, some recruitment to mRNA. Typically, transla- thereby facilitating decapping and degradation tion begins when the 43S ribosomal complex is (Mauer et al. 2017). recruited to the 50 7-methylguanosine (m7G) cap Ψ also affects mRNA stability. In yeast, Pus7p of mRNA transcripts through the cap-binding pseudouridylates mRNA in response to heat complex eukaryotic translation initiation factor shock. Depletion of Pus7p results in decreased 4F (eIF4F). Under stress conditions, a switch stability of theses transcripts, supporting a role from canonical eukaryotic translation initiation for Ψ in mRNA degradation (Schwartz et al. factor 4E (eIF4E)-dependent to eIF4E-indepen- 2014). dent translation initiation can take place (see Translation is also affected by the availability Kwan and Thompson 2018; Merrick and Pavitt of transcripts for binding. Thus, sub- 2018; Robichaud et al. 2018). Toeprinting ex- cellular compartmentalization such as nuclear periments revealed that m6A at the 50UTR of retention controls transcript availability. The ef- mRNA transcripts can allow bypass of eIF4E- fect of m6A on the nuclear export of transcripts dependency through the direct binding of eIF3, was shown by Fustin et al. (2013) who showed which is sufficient to recruit the 43S complex that reduced m6A levels resulted in nuclear ac- to initiate translation (Meyer et al. 2015). These cumulation of otherwise methylated transcripts. experiments showed that eIF3 preferentially Axonal mRNA can be locally translated in binds mRNA transcripts that carry m6A in the response to specific signals, providing an effec- 50UTR, and that m6A-induced translation initi- tive mechanism for rapid protein synthesis. The ation occurs only in transcripts containing m6A nonnuclear pool of FTO in the axon regulates in the 50UTR and not elsewhere within the tran- local translation of axonal mRNA by m6A de- script (including the cap-associated m6Am). methylation to increase transcript expression Another line of evidence for the role of m6A (Yu et al. 2017). in translation initiation came from a study that 5mC can also affect nuclear export of mRNA showed direct binding of METTL3 to eIF3 (Lin transcripts. Depletion of 5mC inhibits recogni- et al. 2016). METTL3, a predominantly nuclear tion of transcripts by the export adaptor ALYREF, protein, is also present in the and an RNA-binding protein that preferentially recruits the ribosome through association with binds 5mC-modified RNA, resulting in dys- eIF3. This association is dependent on m6A regulated nuclear export (Yang et al. 2017a). methylation and independent of METTL3’s methyltransferase activity, METTL14, WTAP, 6 Direct Effects and other m A reader proteins, including YTHDF1 and YTHDF2. Reduced m6A levels The effects of mRNA modifications on transla- lead to decreased METTL3 binding to tran- tion can take place at every stage of the transla- scripts in the cytoplasm and consequently re- tion process. These modifications can act by duced protein levels (Lin et al. 2016). It is either altering base-pairing, inducing confor- unclear whether METTL3 plays a role in cap- mational changes, or modulating the recogni- dependent or cap-independent translation. tion of RNA-binding proteins. Most of the The effect of m6A in the 50UTR on transla- data regarding mRNA modification emerge tion initiation was further established in a recent from m6A studies. Although it does not affect study (Coots et al. 2017) revealing that when hydrogen bonding or translational fidelity (You eIF4F-dependent translation is impaired, cells et al. 2017), accumulating evidence suggests use a different mode of translation that is neither that m6A is involved in translation regulation cap- nor internal ribosome entry site (IRES)- through several mechanisms. dependent, but rather m6A-dependent. This Transcriptome-wide mapping of m6A iden- study identified ATP-binding cassette subfamily tified a subset of m6A sites that are located in the F member 1 (ABCF1) as a critical mediator of 50UTR of mRNA (Dominissini et al. 2012; Me- m6A-dependent, eIF4E-independent translation. yer et al. 2012). The 50UTR is critical for ribo- ABCF1 serves as an alternative recruiter for the

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The Epitranscriptome in Translation Regulation

ternary complex during noncanonical transla- and Ψ, requiring a longer time for synthesis tion by interacting with eIF2, which controls of full-length proteins compared to unmodified ternary complex formation (a rate-limiting step mRNA (Svitkin et al. 2017). to the overall translational output) and ribo- Translation terminates when the ribosome somes, proving its critical role for mRNA trans- reaches a nonsense codon (see Hellen 2018; lation under stress (see Merrick and Pavitt Rodnina 2018). All three possible nonsense 2018; Wek 2018). Interestingly, the synthesis codons contain a uridine at the first position, of METTL3, required for installing m6Ain and Karijolich and Yu (2011) have shown that mRNA transcripts, is m6A- and ABCF1-depen- pseudouridylation of this residue suppresses dent, generating a positive feedback loop and translation termination in vitro and in vivo. providing a mechanism by which cells activate An in-depth structural study of the bacterial m6A-mediated translation on inhibition of cap- 30S ribosomal subunit in complex with a pseu- dependent translation. douridylated nonsense codon indicates forma- The effect of m6A on translation initiation tion of noncanonical base-pairing in the second involves additional m6A-binding proteins. and third positions (Fernandez et al. 2013). Wang et al. (2015) have shown that a member Although it is unclear whether this readthrough of the YTH domain family is involved in m6A- mechanism is used naturally, it could be of mediated regulation of translation initiation. therapeutic importance, given that many genet- YTHDF1 binds m6A-modified transcripts and ic diseases can be attributed to premature increases translation through interaction with translation termination yielding a dysfunctional eIF3 (Wang et al. 2015). protein (Karijolich and Yu 2011). Although the effect of m6A on translation initiation is mediated through its binding by MODIFICATIONS AND INNATE IMMUNITY: different proteins, other modifications may act 0 GLOBAL EFFECTS ON THE TRANSLATION by affecting the structure of the 5 UTR. A pos- MACHINERY itively charged m1A may alter the secondary/ tertiary structure of mRNA around translation Innate immunity, the initial immune response initiation sites by blocking Watson–Crick base- to pathogenic invasion, involves the activation pairing or introducing charge–charge interac- of proteins that distinguish self from nonself by tions. Alternatively, potential binding proteins identification of pathogen-associated molecular may specifically recognize m1A and facilitate patterns (PAMPs). DNA and RNA stimulate translation initiation of methylated transcripts the mammalian innate by acti- in a way analogous to the role of m6A readers of vation of Toll-like receptors (TLRs), retinoic the YTH domain family. Ribosome profiling acid-inducible gene I (RIG-I), and the RNA- and proteomic analyses revealed that m1A activated protein kinase R (PKR) (Nallagatla sites in the 50UTR, but not those in the CDS et al. 2011). or 30UTR, correlated with higher translation Similar to DNA-containing methylated efficiency. The mechanisms by which 50UTR CpG motifs, which do not stimulate TLRs, m1A sites affect translation efficiency are still the modified 5mC, m6A, and Ψ unknown (Dominissini et al. 2016; Lin et al. also ablate TLR activity (Kariko et al. 2005). 2016; Li et al. 2017b). Although 50-triphosphate RNA is a ligand for During translation elongation, m6AinmRNA RIG-I, 50 capped or pseudouridylated RNA is can act as a barrier to tRNA accommodation. not (Hornung et al. 2006; Wang et al. 2010). The presence of an m6A within a codon disrupts Consequently, pseudouridylated in vitro tran- cognate tRNA selection, with the greatest effect scribed RNA shows significantly enhanced trans- at the most thermodynamically unstable steps lation compared to nonmodified RNA, whereas a of initial selection (Choi et al. 2016). 5mC and modest effect was also observed when incorpo- Ψ also affect elongation rates in vitro, with in rating 5mC (Kariko et al. 2008). Interestingly, vitro transcribed mRNAs containing both 5mC enhanced translation was observed in rabbit

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reticulocyte lysates, which contain the RNA- 2014), we and others found that complete dependent PKR, but not in wheat germ extracts, knockout of Mettl3 in mESCs abrogates the which do not (Davis and Watson 1996). ability of mESCs to undergo differentiation (Ba- The improved translation of pseudouridy- tista et al. 2014; Geula et al. 2015). Differentia- lated mRNA is independent of RIG-I activity tion is a cellular process that is hypothesized to and further research showed that in vitro tran- require concerted and timely degradation of scribed RNA-containing uridine activates PKR, pluripotency factors, and depletion of m6A leads which phosphorylates the α subunit of eIF2, to continued expression of pluripotency factors, resulting in inhibition of translation (Dever such as Nanog and Oct4, even as differentiation et al. 1992). Replacing uridine with Ψ diminish- is triggered. The continued expression of the es PKR activation (Anderson et al. 2010). Curi- more stable pluripotent mRNAs, because of ously, in the same setting, incorporation of m6A their reduced m6A levels, results in their higher instead of adenosine in mRNAs rendered them protein levels (Geula et al. 2015). untranslatable (Kariko et al. 2008). Recently, Another tightly controlled process, early Svitkin et al. (2017) showed that N1-methyl-Ψ embryogenesis, requires clearance of maternal outperforms Ψ and several other mRNAs in maternal-to-zygotic transition (MZT) modifications in translation by combining re- (Lee et al. 2014). Zhao et al. (2017) neatly showed duced immunogenicity with altered dynamics the importance of YTHDF2-mediated removal of the translation process. Collectively, these find- of m6A-methylated maternal transcripts for ings reveal a complex interplay between mRNA MZT in zebrafish. modifications, innate immunity, and translation. The cellular response to stress presents an- other system that relies on prompt shifts in ex- 6 DIFFERENTIAL mRNA MODIFICATION pression patterns. When we mapped the m A AFFECTS TRANSLATION IN COMPLEX methylome, we noticed a dynamic response to BIOLOGICAL PROCESSES various stimuli, such as ultraviolet irradiation and exposure (Dominissini et al. Prior to the emergence of high-throughput 2012). Recent studies have examined in greater methods, studies of the m6A methyltransferase depth the role that m6A plays in the cellular METTL3 showed the modification to be neces- response to stress. In response to heat shock sary for elaborate biological processes requiring stress, the m6A reader YTHDF2 undergoes differentiation, such as embryogenesis in Arabi- stress-induced localization to the nucleus, where dopsis thaliana (Zhong et al. 2008) and oogen- it protects 50UTR methylation of pertinent tran- esis in Drosophila melanogaster (Hongay and scripts from demethylation by FTO (Zhou et al. Orr-Weaver 2011). The involvement of m6Ain 2015). The protected m6A residues promote fertility was substantiated when a study of cap-independent translation of heat shock pro- the m6A demethylase ALKBH5 showed that tein 70 (Hsp70) mRNA, providing a mechanism dysregulated m6A levels in mice, as a result of for selective translation under heat shock stress. ALKBH5 knockout, led to defects in spermato- Knockdown of YTHDF2 disrupts this mecha- genesis, possibly because of dysregulated splic- nism, leading to reduced synthesis of Hsp70 ing and mRNA expression (Zheng et al. 2013; after exposure to heat shock stress (Zhou et al. Tang et al. 2017). 2015). Further evidence of this mechanism is Following the mapping of m6A in mamma- found in the work of Meyer et al. (2015), which lian cells, which revealed the extent and conser- describes transcriptome-wide redistribution of vation of the modification, its role in differenti- m6A in response to diverse cellular stresses, ation was explored further. Although the first resulting in an increased number of mRNAs study of m6A in mouse embryonic stem cells with 50UTR methylation capable of promoting (mESCs) found, by knockdown of the methyl- cap-independent translation, including similar transferases Mettl3 and Mettl14, that m6Ais results regarding the translation of Hsp70 fol- required for self-renewal of mESCs (Zhu et al. lowing heat stress.

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The Epitranscriptome in Translation Regulation

More evidence for the role of m6A in regu- anism resulted in cell-cycle arrest and differen- lating translation in response to stress came from tiation of AML cells (Barbieri et al. 2017). researching the role of YTHDF1, where Wang As discussed earlier, m6A also positively et al. (2015) showed that tethering the noncata- regulates expression through the activity of lytic amino terminus of YTHDF1 to a transcript METTL3 in its capacity as an m6A reader. augments its translation during recovery from Increased expression of METTL3 in lung ade- arsenic stress. nocarcinoma promotes growth, survival, and Involvement of m6A in response to stress invasion of tumor cells (Lin et al. 2016). Some also appears to play a part in carcinogenesis. observations of m6A affecting translation are Zhang et al. (2016a,b) found that exposure of still without mechanistic insight. For example, cells to hypoxia results in reduced it has been found that m6A allows a flow of m6A levels in pluripotency factor NANOG information from transcription to translation, and Kruppel-like factor 4 (KLF4) mRNAs as inefficient transcription results in increased because of both inhibited zinc finger protein m6A deposition, causing decreased translation 217 (ZNF217)-dependent methylation and in- efficiency (Slobodin et al. 2017). creased ALKBH5-dependent demethylation, Overall, these findings show that differential thus increasing their protein levels and, subse- methylation, or reading thereof, affects transla- quently, the breast cancer stem-cell phenotype. tion in complex biological processes, and as a In contrast to the decrease in m6A levels in plu- result plays a key role in determining cell fate. ripotency factors in breast cancer cells, exami- Other recently identified mRNA modifications, nation of the global response of HEK293T cells such as m1A (Dominissini et al. 2016) and Ψ to hypoxia revealed a general m6A increase in (Schwartz et al. 2014), have been reported to poly(A) RNAs, conferring increased mRNA be dynamic in response to stress. Deciphering stability and improved recovery of translational the roles that they play in translational regula- efficiency (Fry et al. 2017). tion promises to be an interesting avenue of Reduction in m6A levels has also been found research. to promote the tumorigenicity and self-renewal of glioblastoma stem-like cells (GSCs) because CONCLUDING REMARKS of altered mRNA expression of key genes (Cui et al. 2017). In addition, increased ALKBH5 ex- Research on the epitranscriptome has quickly pression is seen in GSCs, where it demethylates moved from one of identification and mapping transcripts of the transcription factor Forkhead to the investigation of function and of the Box M1 (FOXM1), thereby enhancing its expres- biological implications of mRNA modifications. sion, thus revealing a new pathway for GSC pro- Studies of the role that these modifications play liferation and tumorigenesis (Zhang et al. 2017). in translation paint a complex picture. When In acute myeloid leukemia (AML) a differ- examining direct regulation of translation, it ent m6A demethylase plays an oncogenic role, appears that the same modification can either although interestingly, by negatively regulating promote or repress translation of an mRNA, genes important for normal hematopoiesis and depending on the location of the modification differentiation. Unlike the role of ALKBH5 in or the biological system studied. Indirectly, the breast cancer or glioblastoma, FTO promotes same modification can either increase mRNA leukemogenesis by demethylating target genes, stability, as in the case of hypoxia-induced leading to decreased protein levels (Li et al. stabilization of mRNA (Fry et al. 2017), or de- 2017c). In line with this result, METTL3 plays crease it, as exemplified in the widely studied an oncogenic role in AML by methylating YTHDF2-mediated decay of m6A-methylated coding regions of genes necessary for leukemo- mRNA (Wang et al. 2014; Du et al. 2016). These genesis, including the SP1. This meth- contrasting effects ultimately have different con- ylation relieves ribosome stalling and leads to sequences for translation output. With m6A, the increased translation. Disruption of this mech- most studied modification, we are now begin-

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ning to understand that the delicate balance of Chekulaeva M, Rajewsky N. 2018. Roles of long noncoding methylation and demethylation is involved in RNAs and circular RNAs in translation. Cold Spring Harb Perspect Biol doi: 10.1101/cshperspect.a032680. complex biological processes such as differenti- Choi J, Ieong KW, Demirci H, Chen J, Petrov A, Prabhakar ation (Geula et al. 2015) and the stress response A, O’Leary SE, Dominissini D, Rechavi G, Soltis SM, et al. (Zhou et al. 2015). Alterations to that balance 2016. N6-methyladenosine in mRNA disrupts tRNA contribute to different pathologies (Li et al. selection and translation-elongation dynamics. Nat Struct Mol Biol 23: 110–115. 2017c; Zhang et al. 2017). The epitranscriptome, fi Cohn WE. 1951. Some results of the applications of ion- consisting of various RNA modi cations, is thus exchange chromatography to nucleic acid chemistry. beginning to be unraveled as a complex layer of J Cell Comp Physiol 38: 21–40. information with major implications for the Coots RA, Liu XM, Mao Y, Dong L, Zhou J, Wan J, Zhang X, 6 regulation of translation in healthy and disease Qian SB. 2017. m A facilitates eIF4F-independent mRNA translation. Mol Cell doi: 10.1016/j.molcel.2017.10.001. states. 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Cite this article as Cold Spring Harb Perspect Biol 2019;11:a032623 13 Downloaded from http://cshperspectives.cshlp.org/ on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press

The Epitranscriptome in Translation Regulation

Eyal Peer, Sharon Moshitch-Moshkovitz, Gideon Rechavi and Dan Dominissini

Cold Spring Harb Perspect Biol 2019; doi: 10.1101/cshperspect.a032623 originally published online July 23, 2018

Subject Collection Translation Mechanisms and Control

Protein Synthesis and Translational Control: A Principles of Translational Control Historical Perspective John W.B. Hershey, Nahum Sonenberg and Soroush Tahmasebi, Nahum Sonenberg, John Michael B. Mathews W.B. Hershey, et al. Translational Control in the Brain in Health and The Epitranscriptome in Translation Regulation Disease Eyal Peer, Sharon Moshitch-Moshkovitz, Gideon Wayne S. Sossin and Mauro Costa-Mattioli Rechavi, et al. Phosphorylation and Signal Transduction Translational Control in Cancer Pathways in Translational Control Nathaniel Robichaud, Nahum Sonenberg, Davide Christopher G. Proud Ruggero, et al. Translational Control during Developmental Roles of Long Noncoding RNAs and Circular Transitions RNAs in Translation Felipe Karam Teixeira and Ruth Lehmann Marina Chekulaeva and Nikolaus Rajewsky Stress Granules and Processing Bodies in Ribosome Profiling: Global Views of Translation Translational Control Nicholas T. Ingolia, Jeffrey A. Hussmann and Pavel Ivanov, Nancy Kedersha and Paul Anderson Jonathan S. Weissman Fluorescence Imaging Methods to Investigate Noncanonical Translation Initiation in Eukaryotes Translation in Single Cells Thaddaeus Kwan and Sunnie R. Thompson Jeetayu Biswas, Yang Liu, Robert H. Singer, et al. Translational Control in Virus-Infected Cells Mechanistic Insights into MicroRNA-Mediated Noam Stern-Ginossar, Sunnie R. Thompson, Gene Silencing Michael B. Mathews, et al. Thomas F. Duchaine and Marc R. Fabian Nonsense-Mediated mRNA Decay Begins Where Toward a Kinetic Understanding of Eukaryotic Translation Ends Translation Evangelos D. Karousis and Oliver Mühlemann Masaaki Sokabe and Christopher S. Fraser

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