Chemical Modifications to RNA: A New Layer of Gene Expression

In contrast to the well-established roles of 5-methylcytosine for epigenetic regulation of gene expression, the functional roles of N6-mA remain elusi...
0 downloads 7 Views 978KB Size
Download PDF
Reviews pubs.acs.org/acschemicalbiology

Chemical Modifications to RNA: A New Layer of Gene Expression Regulation Jinghui Song† and Chengqi Yi*,†,‡ †

State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, and Peking-Tsinghua Center for Life Sciences and ‡Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China ABSTRACT: The first chemical modification to RNA was discovered nearly 60 years ago; to date, more than 100 chemically distinct modifications have been identified in cellular RNA. With the recent development of novel chemical and/or biochemical methods, dynamic modifications to RNA have been identified in the transcriptome, including N6-methyladenosine (m6A), inosine (I), 5-methylcytosine (m5C), pseudouridine (Ψ), 5-hydroxymethylcytosine (hm5C), and N1-methyladenosine (m1A). Collectively, the multitude of RNA modifications are termed epitranscriptome, leading to the emerging field of epitranscriptomics. In this review, we primarily focus on recently reported chemical modifications to mRNA; we discuss their chemical properties, biological functions, and mechanisms with an emphasis on their high-throughput detection methods. We also envision that future tools, particularly novel chemical biology methods, could further facilitate and enable studies in the field of epitranscriptomics.

B

enzymes, high-throughput detection methods, and biological functions.

esides the four regular nucleosides (adenosine, guanosine, cytidine, and uridine), natural RNA molecules contain over 100 chemically modified nucleosides in the three kingdoms of life (http://mods.rna.albany.edu/mods/). Most RNA modification studies have focused on the abundant noncoding RNAs (ncRNA), such as rRNA, tRNA, and small nuclear RNA (snRNA). These studies reveal that RNA modifications are important for the functions of these ncRNAs in translation and splicing.1−3 Messenger RNA (mRNA) and long noncoding RNA (lncRNA) are also decorated by modifications, such as the 5′ cap and RNA editing. In eukaryotes, the 5′ cap, which is a 7-methylguanosine linked to the 5′ terminus of mRNA (and some lncRNA) via an unusual 5′ to 5′ triphosphate linkage, functions importantly in RNA stability and translation.4,5 RNA editing can change the coding message in mRNA and diversify protein synthesis.6,7 Benefiting from recently developed high-throughput detection strategies, diverse chemical modifications have been identified in mRNA, including N6-methyladenosine (m6A), inosine (I), 5-methylcytosine (m5C), pseudouridine (Ψ), 5hydroxymethylcytosine (hm5C), and N1-methyladenosine (m1A). The multitude of dynamic modifications in mRNA have diverse biological functions. Understanding the distribution, mechanisms, regulation, and function of these dynamic RNA modifications will greatly expand our knowledge of epitranscriptomics. Because inosine has been reviewed extensively elsewhere,8,9 we will focus on five recently identified chemical modifications (m6A, m5C, hm5C, Ψ, and m1A) in mRNA. We will discuss their chemical properties, modification © 2017 American Chemical Society

■

CHEMICAL PROPERTIES AND BIOGENESIS OF RNA MODIFICATIONS As the most prevalent internal modification in all higher eukaryote mRNA and lncRNA, m6A (Figure 1) was discovered in the 1970s and occurs on an average of ∼3 sites per mRNA molecule.10 The methyl group of m6A can exist in both syn and anti conformations: although the syn conformation is more energetically favored than the anti conformation in singlestranded RNA (ssRNA),11,12 the methyl group in m6A must adopt a high-energy anti orientation in paired RNA to accommodate normal Waston−Crick base pairs.13 Thus, N6 methylation in m6A could destabilize the base pairs of its local RNA regions.13,14 The formation of m6A is catalyzed by “writers”, which is a methyltransferase complex consisting of methyltransferase like 3 (METTL3), methyltransferase like 14 (METTL14), and regulatory subunit Wilms’ tumor 1associating protein (WTAP).15−19 In the complex, METTL3 serves as the catalytically active subunit, METTL14 enhances substrate recognition and RNA binding, and WTAP may facilitate the translocation into nuclear speckles.19−21 Biochemical analyses have revealed that the consensus substrate Received: October 31, 2016 Accepted: January 4, 2017 Published: January 4, 2017 316

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology

Figure 1. Chemical structures of m6A, m1A, m5C, hm5C, and Ψ.

Figure 2. Dynamic RNA modifications in mRNA and lncRNA affect a wide range of cellular processes. In the nucleus, RNA modifications affect premRNA and pri-miRNA processing and nuclear export. In the cytoplasm, RNA modifications regulate mRNA translation and stability. Red and black lines represent RNA and DNA, respectively. RNA modifications are indicated by blue filled circles.

sequence of the m6A methyltransferase is RRm6ACH (R= A, G; H = A, C, U).22 m6A can be demethylated by “erasers”, fat mass and obesity-associated protein (FTO) and alkylation repair homologue protein 5 (ALKBH5), which belong to the Fe(II)dependent and 2-oxoglutarate-dependent oxygenase superfamily.23,24 FTO oxidatively removes m6A modification through intermediates N6-hydroxymethyladenosine (hm6A) and N6formyladenosine (f6A).25 However, hm6A and f6A have not been detected in the demethylation process by ALKBH5.26 The discovery of the “erasers” sheds light on the reversible nature of m6A, indicating its potential regulatory function. In addition to methylation at the N6 position, it can also occur at the N1 position of adenosine, forming m1A (Figure 1). m1A was first discovered in the 1960s27,28 and is prevalent in tRNA and rRNA where it plays a role in maintaining tRNA tertiary structure, ribosome biogenesis, and translation.29−31 Recent studies report that m1A is also a dynamic and prevalent modification in mRNA.32,33 Notably, the N1 methyl group in m1A can introduce a positive charge under physiological conditions; thus, it influences the strength of hydrogen bonds and facilitates the interaction with negatively charged phosphates in the backbone.34−36 Furthermore, because of the methyl group in the N1 position of m1A at the Watson− Crick interface, m1A can interfere with normal base pairing.37 In 28S rRNAs of human and mouse cells, m1A is modified by nucleomethylin (NML; also known as RRP8);38 in human tRNA, m1A formation is catalyzed by TRMT61B, TRMT10C, and the complex of TRMT6 and TRMT61A.39−41 Very recently, m1A in tRNA was reported to be reversible by ALKBH1 in mammalian cells;42 m1A in mRNA could also be targeted by ALKBH3.32,33

m5C is another methylation in mRNA (Figure 1). Previously, it has been reported that m5C exists in highly abundant tRNA and rRNA in both prokaryote and eukaryote organisms,43 where it stabilizes the RNA secondary structure and affects translational fidelity.34,44−46 Through recent high-throughput strategies, m5C has been found in mRNA and other ncRNA.47−50 The formation of m5C is catalyzed by the methyltransferases DNMT2 and NSUN2 in human cells.47−49 Although m5C has not been reported to be reversible in RNA, it can be further oxidized to hm5C (Figure 1) by ten-eleven translocation (TET) family enzymes,51−53 suggesting that m5C may be regulated. The discovery of m5C and hm5C in mRNA further expand the alphabet of RNA epigenetics and make epitranscriptome more intricate and diverse. Modifications in RNA are diverse, and the chemical functionalities can certainly go beyond methylation. Known as the “fifth nucleotide” of RNA, Ψ (Figure 1) is a C−C glycosidic rotation isomer of uridine, which was first found in 1951 in RNA.54,55 Although Ψ has the same molecular weight and base pairing pattern with regular U, an additional H-bond donor in the N1H of Ψ can bind a water molecule to bridge the interactions of this N1H and the preceding phosphate groups and stabilize the RNA structure.56 Ψ is the most abundant RNA modification, present in tRNA, rRNA, and snRNA; until recently, it had not been found in mRNA and lncRNA.57−61 Ψ plays important roles in folding and translation fidelity of rRNA, stability and decoding of tRNA, function of snRNA, and splicing and translation of mRNA.3 There are two pathways for the formation of Ψ in eukaryotes: one is catalyzed by the RNAdependent pseudouridine synthases (PUSs) that require the cofactor box H/ACA ribonucleoproteins as guides to recognize different substrates; another is catalyzed by the RNA317

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology independent PUSs that require no cofactor.57,62,63 Ten PUSs were identified in yeast: Cbf5 is an RNA-dependent PUS, and the rest (Pus1−9) are RNA independent.64 Thirteen PUSs were identified in human: only DKC1 (a homologue of Cbf5) is an RNA-dependent PUS.57 The study of Ψ is complicated by multiple PUSs and potential functional redundancy. The inert C−C glycosidic bond of Ψ makes it difficult to be reversible. Interestingly, Ψ can be further methylated to 1-methylpseudouridine (m1Ψ) by EMG1,65 potentially offering a mechanism to reduce the level of Ψ. The abundance of m6A, m1A, m5C, hm5C, and Ψ in mRNA are quite different as quantified by the same method (LC-MS/ MS). As the most abundant internal mRNA modification in eukaryotes, the m6A/A ratio of human mRNA is approximately 0.4−0.7%.16,23,24 The ratio of Ψ/U in mammalian mRNA is approximately 0.2−0.6%, which is comparable in content to m6A.61 Thus, m6A and Ψ are two widespread chemical modifications in mRNA. In human mRNA, the m5C/C ratio is approximately 0.02−0.09%, and the hm5C/C ratio is approximateyl 0.001−0.003%, only 1/25 of m5C.52 The m1A/ A ratio in mammalian mRNA is approximately 0.02− 0.06%,32,33 approximately 1/10 of m6A. Among the five modifications, m6A and Ψ are modifications with high abundance in mRNA, and hm5C is the one with the lowest abundance.

decay (Figure 2). On the other hand, the m6A mark can also affect the translation efficiency of mRNA. It has been reported that the translation efficiency was increased modestly upon METTL3 knockout in mouse embryonic stem cells (mESCs) and embryoid bodies (EBs), suggesting a negative regulatory role of m6A in translation.74 However, when m6A is recognized by another cytoplasmic m6A reader YTHDF1 (which interacts with initiation factors and ribosomes), these m6A-marked transcripts are preferentially translated.75 More recently, it has been reported that m6A in the 5′ UTR of mRNA can promote cap-independent translation initiation.76,77 The researchers further found that eukaryotic initiation factor 3 (eIF3) directly binds m6A-containing 5′ UTR and recruits the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E.76 These data provide direct evidence for a function of m6A in translational regulation (Figure 2), although METTL3 can also directly promote translation independently of its catalytic activity and m6A reader proteins.78 In addition, the regulatory role of m6A in gene expression has been reported to affect ESC pluripotency in human and mouse.17,74,79−82 Very recently, it was also found that m6A is required for XISTmediated transcriptional repression of genes on the X chromosome.83 Besides the functions in host cells, m6A was also found in viral RNA genomes. Several groups independently reported the function of m6A during virus infection. It has been reported that m6A in both host and viral mRNAs were increased during HIV-1 infection of human CD4 T cells.84 The researchers further found that m6A in the stem loop II region of HIV-1 Rev response element (RRE) RNA modulates the interaction between Rev protein and RRE RNA and influences the nuclear export of RNA. Moreover, one study found that binding of YTHDF1−3 proteins to m 6 A-methylated HIV-1 RNA promotes viral protein and RNA expression,85 whereas another recent study found that YTHDF1−3 proteins bind m6Amodified HIV-1 RNA and inhibit viral reverse transcription (RT).86 Very recently, m6A has also been found in the RNA genomes of flavivirus Zika (ZIKV), hepatitis C virus (HCV), and other Flaviviridae family members and modulates infection by the Flaviviridae virus.87,88 These data suggested the important role of m6A in viral infection. Given that cells can discriminate self and nonself by RNA modifications,89−92 RNA modifications may globally participate in immune response. In addition to m6A, Ψ in mRNA might also affect mRNA splicing, stability, and translation (Figure 2). First, the replacement of U with Ψ in the polypyrimidine tract (near the 3′ splice site) of artificial pre-mRNA inhibits pre-mRNA splicing by affecting U2 auxiliary factor (U2AF) binding in Xenopus oocytes.93 However, whether or not Ψ is present in pre-mRNA remains to be investigated. Second, one study in yeast has demonstrated heat shock-induced Ψ sites. Interestingly, the expression level of the heat shock induced, Pus7dependent pseudouridylated transcripts are higher in WT yeast than in Pus7-deletion yeast, suggesting that Ψ could contribute to RNA stability.58 Third, pseudouridylation appears to impact mRNA translation in a complicated manner. When Ψcontaining mRNA was translated in rabbit reticulocyte lysates, the presence of Ψ stimulates translation. However, in wheat germ extracts and bacterial cell lysates, Ψ represses translation.94 When Ψ was incorporated in different positions and codons of mRNA, different effects on translation were also observed. One study has reported that the incorporation of a single Ψ at each position of the codon “UUU” of bacterial

■

BIOLOGICAL FUNCTIONS With the development of high-throughput technologies of m6A, more and more functions of m6A have been discovered in a wide range of cellular processes. For instance, right after transcription, processing of pre-mRNA and primary microRNAs (pri-miRNAs) are critical steps to generate mature mRNA and miRNA. Many investigations have provided strong evidence for the regulatory role of m6A in pre-mRNA and primiRNA processing (Figure 2). For pri-miRNA processing, m6A is present in pri-miRNA and promotes miRNA biogenesis in which HNRNPA2B1 is involved.66,67 For pre-mRNA processing, m6A can alter its RNA local structure to regulate the interactions of RNA and HNRNPC, which can further affect the alternative splicing of m6A-containing mRNAs mediated by HNRNPC.68 YTHDC1 is a nuclear reader of m6A that can directly regulate inclusion of targeted exons through recruiting SRSF3 while blocking the binding of SRSF10 to mRNA.69 The increased m6A level could also promote the RNA binding ability of SRSF2, leading to increased inclusion of target exons.70 Besides splicing, m6A may affect alternative polyadenylation (APA) as well.71,72 After pre-mRNA processing in the nucleus, it is crucial that mature mRNAs are exported to the cytoplasm. Researchers have found that nuclear mRNA export is accelerated upon ALKBH5 knockdown, indicating that m6A might be involved in mRNA export (Figure 2).24 After export out of nucleus, mature mRNA can be either translated or degraded in the cytoplasm. mRNA turnover is an important and fast process that regulates mRNA abundance, allowing for rapid cellular adjustment in various environments. Researchers found that adjacent m6A methylation might block the binding of the human antigen R (HuR) to destabilize mRNA.17 Moreover, YTH domain family 2 (YTHDF2, the first comprehensively established m6A reader) selectively binds to m6A-decorated mRNA to promote the decay of thousands of mRNAs via the translocation of bound mRNA from the translatable pool to processing bodies.73 These results revealed the tight regulation between m6A modification and mRNA 318

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology

Figure 3. Dimroth rearrangement and demethylase can eliminate the RT signatures of m1A. (a) m1A in RNA causes stop or misincorporation during RT, whereas the conversion of m1A to m6A or regular A can remove the RT signatures of m1A. (b) Dimroth rearrangement of m1A under alkaline conditions. (c) Demethylation of m1A by AlkB.

containing RNA for high-throughput sequencing. These approaches were quickly adopted by many other laboratories and are used frequently today. Such methods have identified thousands of m6A peaks in mammalian mRNA and lncRNA: the m6A peaks are enriched in the 3′ UTR and near stop codons. Moreover, m6A is also a widespread modification in other species mRNA: in yeast, m6A is dynamically regulated methylation during meiosis;101 in plants, m6A peaks are abundant around the start codon (16%).102 To reach singlebase-resolution m6A methylomes and quantify the m6A stoichiometry, several groups have independently reported the optimized strategies based on the m6A/MeRIP-seq protocols. In m6A-CLIP and miCLIP strategies, 254 nm UV light was used to induce covalent cross-links between antibody and m6A-containing RNA.72,103 During RT, the antibody−RNA cross-linking sites can cause the mutational or truncational signatures of cDNA so that base-resolution m6A sites can be identified. In PA-m6A-seq, photoreactive nucleoside analogue (4-thiouridine, 4SU), which can improve the cross-linking efficiency, was added to the growth medium and then incorporated into newly synthesized RNA molecules; after immunoprecipitation, covalent cross-links between antibody and RNA were formed under UV irradiation at 365 nm (instead of UV 254 nm).104 Notably, the incorporated 4SU can lead to T to C transition at cross-linking sites; thus, it is possible to identify m6A at base-resolution. Recently, a new modified strategy, called m6A-LAIC-seq, was developed to quantify the m6A stoichiometry on a transcriptome-wide scale.71 It revealed that most genes exhibit less than 50% m6A modification levels in human ESC. The development of optimized detection methods would help us to better understand the intricate m6A methylome.

mRNA represses translation in vitro with the strongest repression at the third codon position.95 In a second study, the replacement of U with Ψ at stop codons suppresses translation termination and converts nonsense codons into sense codons both in vitro and in vivo.96,97 However, all the experiments regarding translation were performed using artificial mRNA carrying Ψ. Whether natural Ψ in mRNA could also affect translation in vivo is worth further investigation. In addition to m 6 A and Ψ, other internal mRNA modifications might affect mRNA translation as well (Figure 2).98 For instance, a single m5C in mRNA could reduce ∼40% of the amount translation product using bacterial whole cell extract, and m5C at the second nucleotide of the codon “CCC” could induce recoding.95 Additionally, uncapped mRNAs that are randomly incorporated by 50% m5C significantly decrease translation in HeLa cell extracts.76 In contrast to m5C, which negatively impacts translation, hm5C is shown to associate with active translation in Drosophila.53 Furthermore, m1A might also contribute to translation. It has been reported that the genes carrying m1A around the start codon correlate with higher protein level, indicating the positive effect of m1A on translation.33 All these phenomena favor the regulatory role of internal mRNA modifications on translation.

■

HIGH-THROUGHPUT DETECTION METHODS The first transcriptome-wide m6A maps with resolution of 100−200 nt were generated in 2012 by two independent groups based on an m6A-specific antibody (m6A/MeRIPseq).99,100 Because of the lack of chemical methods to differentiate m6A from A, these approaches used an m6Aspecific antibody to pull down m6A-containing RNA (but not non-m6A RNA), which allows pre-enrichment of m6A319

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology

Figure 4. Bisulfite-mediated conversion of cytosine to uracil. (a) Principle of RNA bisulfite sequencing. (b) Cytosine deamination via bisulfite treatment.

Figure 5. CMC specifically labels Ψ. (a) Work flow of CMC labeling. (b) Chemical structure of CMC. (c) CMC acylation of U, Ψ, and G. (d) After alkaline treatment, only the adducts of CMC-Ψ remain.

Unlike m6A, which still pairs to thymidine during RT, m1A can interfere with Watson−Crick base pairing. Recently, we and others independently developed two approaches (called m1AID-seq and m1A-seq) to map the m1A methylomes.32,33 Both approaches are based on m1A-specific antibody, which permits the pre-enrichment of m1A-containing RNA. After immunoprecipitation, the m1A-containing RNA was subjected to an additional treatment step to remove the RT signatures of m1A. Therefore, by comparing the sequencing profiles of treated and untreated samples, high-confidence m1A peaks can be identified. In particular, m1A-seq exploited Dimroth rearrangement to convert m1A to m6A under alkaline conditions because m6A neither changes the base-pairing properties nor inhibits RT;105 the conversion of m1A to regular A by demethylase was used in m1A-ID-seq to eliminate the RT signatures of m1A (Figure 3). Such methods first report that m1A is a new, dynamic, and reversible epitranscriptomic mark in mammalian mRNA and lncRNA, which is enriched in the 5′ UTR.

The detection of m5C in RNA could draw lessons from m dC detection in DNA for which bisulfite treatment is widely used. A modified bisulfite strategy, which converts unmodified cytosines (but not m5C) to uracils (Figure 4), was reported to develop m5C sequencing in RNA.106 In 2012, the first profile of m5C was obtained by coupling bisulfite conversion with highthroughput sequencing, indicating the presence of m5C in human mRNA and lncRNA.47 Several groups have also made efforts to develop new approaches to identify m5C sites.48−50 Aza-IP exploited the formation of 5-azacytidine-methyltransferase adducts to enrich and subsequently sequence m5C targets; this approach was applied to identify the direct targets of NSUN2 and DNMT2 in the human tanscriptome. In miCLIP of m5C (different from miCLIP of m6A), the mutation (C271A) of NSUN2, which could form a stable covalent bond with RNA, was exploited to identify the transcriptomewide targets of NSUN2. In the m5C-RIP approach, an antibody against m5C was used to validate m5C sites in archaea mRNA, 5

320

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology but m5C-RIP has not been applied to mammalian cells as yet. Such an antibody-based approach could in principle be applied to other modifications as well. For instance, a method utilizing a hm5C-antibody (called hMeRIP-seq) was reported to identify more than 3,000 hm5C peaks in the Drosophila transcriptome by using an hm5C specific antibody.53 In Drosophila S2 cells, hm5C presents preferentially in polyadenylated RNA and is not detected in rRNA or small RNA by dot blotting. The transcriptome-wide mapping of Ψ sites relies on the chemical N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide (CMC), which specifically labels Ψ (Figure 5).58−61,107 Initially, CMC can acylate guanosine (G) at the N1 position, uridine (U) at the N3 position, and Ψ at the N1 and N3 positions in RNA (Figure 5c); subsequently, the adducts of CMC-G and CMC-U are hydrolyzed under alkaline treatment (pH 10.4), whereas CMC remains specifically at the N3 position of Ψ (Figure 5d).108 The formation of CMC-Ψ adducts can cause stops at one nucleotide 3′ to the labeled Ψ sites during RT, thereby achieving base-resolution detection of Ψ (Figure 5a). Three independent CMC-based profiling methods of Ψ (called Ψ-Seq, Pseudo-Seq, and PSI-Seq) have identified approximately 50−100 Ψ sites in yeast mRNA and 100−400 sites in human mRNA at single-base resolution.58−60 However, these methods cannot pre-enrich Ψ-containing RNA of interest. We developed a chemical pull-down method for Ψ, termed CeU-Seq, which allows pre-enrichment of Ψ-containing RNA as well as Ψ detection at single-base resolution.61 In CeUSeq, CMC derivative azido-CMC (N3-CMC) was used to label Ψ, and the N3-CMC-Ψ adduct can be coupled with biotin via “click” chemistry.109 Then, the Ψ-containing RNA was pulled down through biotin and subsequently sequenced. Through CeU-Seq, we identify over 2,000 Ψ sites in human mRNA and ∼1,500−1,700 Ψ sites in mouse mRNA.

cytosine modifications are resistant to bisulfite treatment, both of which result in false positives.110−112 Although other approaches (Aza-IP and miCLIP of m5C) are bisulfite-free and can pre-enrich m5C targets, overexpression of methyltransferase is required, which may lead to some nonspecific targets due to the high expression and potential mis-localization of the enzyme within the cell. More sensitive and accurate m5C detection methods need to be developed in the future. For Ψ detection, CMC-based Ψ sequencing methods have successfully identified Ψ sites in the transcriptome at single-base resolution, but the alkaline treatment step gives rise to RNA degradation. Further improvements in future Ψ profiling are needed, e.g., finding new chemicals that specifically react with Ψ. For hm5C and m1A detection, current sequencing technologies have not reached single-base resolution, which hinders functional studies of RNA modification. Learning from the success of single-base and quantitative m6A sequencing technologies, optimized sequencing methods of hm5C and m1A are expected in the future. Other considerations are also required for functional studies of RNA modifications. First, it is important to quantify the absolute stoichiometry of RNA modifications. Modifications with high stoichiometry in a certain transcript are more likely to affect the fate of the RNA. At present, except for m6A-LAICseq, most sequencing technologies cannot quantify the percentage of transcripts containing modifications. Second, it is still unknown whether any correlations exist among different RNA modifications. Multiple RNA modifications may together contribute to regulate the biological consequences for a certain transcript. Therefore, it will be helpful to develop highthroughput technologies that can identify multiple modifications at the same molecule simultaneously. Third, all existing RNA modification sequencing methods cannot be applied to single cells directly. It is known that m6A has a regulatory role in ESC pluripotency; thus, single-cell sequencing of m6A, as well as other modifications, is desired to uncover the contributions of RNA modifications in cell fate determination during development and differentiation. In summary, sequencing technologies of RNA modifications are just beginning. We envision that future tools, particularly novel chemical biology methods, could be applied to further facilitate studies in the field of epitranscriptomics.

■

EXISTING CHALLENGES FOR DETECTION OF RNA MODIFICATIONS From the discussions above, we could briefly divide sequencing technologies of RNA modifications into two categories: One is antibody-based, which allows the pre-enrichment to increase the signal-to-noise ratio. The other is chemical labeling-based, which generally enables detection of RNA modifications at single-base resolution. Each category has certain advantages over the other, and the two categories are not mutually exclusive. Major concerns for detecting RNA modifications are the precision, sensitivity, quantitative ability, and resolution of high-throughput detection methods, and many challenges remain to be solved. For m6A detection, single-base-resolution and quantitative detection technologies of m6A have been developed to facilitate the functional studies, but all these sequencing technologies are antibody-based. Indeed, it has been shown that the m6A-specific antibody could have intrinsic bias on RNA sequences and secondary structures and could not distinguish m6A from N6,2′O-dimethyladenosine (m6Am);101,103 therefore, antibody-independent methods are still desired to dissect the distribution of m6A in an unbiased way. For m5C detection, RNA bisulfite sequencing can directly detect site-specific endogenous m5C sites, but it still has several disadvantages: It did not pre-enrich m5C sites of interest, so extremely high depths of sequencing are required to detect methylated RNAs of low abundance. In addition, bisulfite treatment could lead to significant RNA degradation owing to harsh chemical and thermal conditions; incomplete conversion of cytosines may be included, and other

■

CONCLUDING REMARKS Great progress on RNA modifications has been achieved over the past five years due to the rapid development of novel RNA modification sequencing technologies. m6A has been revealed to affect a broad set of biological functions, including mRNA splicing, export, translation, stability, structure, and miRNA biogenesis; emerging evidence also suggests m6A as a regulator in embryonic stem cell state transition and immune response. In recent studies, other dynamic chemical modifications of mRNA (m5C, Ψ, hm5C, and m1A) were also identified in the transcriptome. However, the biological function of these mRNA modifications is poorly understood due to the lack of optimized detection methods and necessary knowledge. First, similar to that of m6A, optimized detection methods are needed to reach single-base resolution and to quantify the stoichiometry. Furthermore, identifying the specific modification enzymes for a given RNA modification is urgent. For m1A, it is still unclear which methyltransferase could catalyze the formation of m1A in mRNA. For Ψ and m5C, modification enzymes are shared between mRNA and tRNA, which make it 321

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology

(6) Fossat, N., Tourle, K., Radziewic, T., Barratt, K., Liebhold, D., Studdert, J. B., Power, M., Jones, V., Loebel, D. A., and Tam, P. P. (2014) C to U RNA editing mediated by APOBEC1 requires RNAbinding protein RBM47. EMBO Rep. 15, 903−910. (7) Nishikura, K. (2016) A-to-I editing of coding and non-coding RNAs by ADARs. Nat. Rev. Mol. Cell Biol. 17, 83−96. (8) Slotkin, W., and Nishikura, K. (2013) Adenosine-to-inosine RNA editing and human disease. Genome Med. 5, 105. (9) Wulff, B. E., Sakurai, M., and Nishikura, K. (2011) Elucidating the inosinome: global approaches to adenosine-to-inosine RNA editing. Nat. Rev. Genet. 12, 81−85. (10) Desrosiers, R., Friderici, K., and Rottman, F. (1974) Identification of methylated nucleosides in messenger RNA from Novikoff hepatoma cells. Proc. Natl. Acad. Sci. U. S. A. 71, 3971−3975. (11) Engel, J. D., and von Hippel, P. H. (1974) Effects of methylation on the stability of nucleic acid conformations: studies at the monomer level. Biochemistry 13, 4143−4158. (12) Engel, J. D., and von Hippel, P. H. (1978) Effects of methylation on the stability of nucleic acid conformations. Studies at the polymer level. J. Biol. Chem. 253, 927−934. (13) Roost, C., Lynch, S. R., Batista, P. J., Qu, K., Chang, H. Y., and Kool, E. T. (2015) Structure and thermodynamics of N6methyladenosine in RNA: a spring-loaded base modification. J. Am. Chem. Soc. 137, 2107−2115. (14) Spitale, R. C., Flynn, R. A., Zhang, Q. C., Crisalli, P., Lee, B., Jung, J. W., Kuchelmeister, H. Y., Batista, P. J., Torre, E. A., Kool, E. T., and Chang, H. Y. (2015) Structural imprints in vivo decode RNA regulatory mechanisms. Nature 519, 486−490. (15) Bokar, J. A., Rath-Shambaugh, M. E., Ludwiczak, R., Narayan, P., and Rottman, F. (1994) Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex. J. Biol. Chem. 269, 17697−17704. (16) Liu, J., Yue, Y., Han, D., Wang, X., Fu, Y., Zhang, L., Jia, G., Yu, M., Lu, Z., Deng, X., Dai, Q., Chen, W., and He, C. (2014) A METTL3-METTL14 complex mediates mammalian nuclear RNA N6adenosine methylation. Nat. Chem. Biol. 10, 93−95. (17) Wang, Y., Li, Y., Toth, J. I., Petroski, M. D., Zhang, Z., and Zhao, J. C. (2014) N6-methyladenosine modification destabilizes developmental regulators in embryonic stem cells. Nat. Cell Biol. 16, 191− 198. (18) Schwartz, S., Mumbach, Maxwell, R., Jovanovic, M., Wang, T., Maciag, K., Bushkin, G. G., Mertins, P., Ter-Ovanesyan, D., Habib, N., Cacchiarelli, D., Sanjana, Neville, E., Freinkman, E., Pacold, Michael, E., Satija, R., Mikkelsen, Tarjei, S., Hacohen, N., Zhang, F., Carr, Steven, A., Lander, Eric, S., and Regev, A. (2014) Perturbation of m6A Writers Reveals Two Distinct Classes of mRNA Methylation at Internal and 5′ Sites. Cell Rep. 8, 284−296. (19) Ping, X. L., Sun, B. F., Wang, L., Xiao, W., Yang, X., Wang, W. J., Adhikari, S., Shi, Y., Lv, Y., Chen, Y. S., Zhao, X., Li, A., Yang, Y., Dahal, U., Lou, X. M., Liu, X., Huang, J., Yuan, W. P., Zhu, X. F., Cheng, T., Zhao, Y. L., Wang, X., Rendtlew Danielsen, J. M., Liu, F., and Yang, Y. G. (2014) Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 24, 177− 189. (20) Wang, X., Feng, J., Xue, Y., Guan, Z., Zhang, D., Liu, Z., Gong, Z., Wang, Q., Huang, J., Tang, C., Zou, T., and Yin, P. (2016) Structural basis of N(6)-adenosine methylation by the METTL3METTL14 complex. Nature 534, 575−578. (21) Wang, P., Doxtader, K. A., and Nam, Y. (2016) Structural Basis for Cooperative Function of Mettl3 and Mettl14 Methyltransferases. Mol. Cell 63, 306−317. (22) Harper, J. E., Miceli, S. M., Roberts, R. J., and Manley, J. L. (1990) Sequence specificity of the human mRNA N6-adenosine methylase in vitro. Nucleic Acids Res. 18, 5735−5741. (23) Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y. G., and He, C. (2011) N6methyladenosine in nuclear RNA is a major substrate of the obesityassociated FTO. Nat. Chem. Biol. 7, 885−887.

difficult to study the function in mRNA. Moreover, identifying the reader proteins that specifically recognize a given RNA modification will contribute further to our understanding of the functions of RNA modifications. We are still at the very beginning of the epitranscriptomics epoch, and we believe that vital input from the chemical biology field will significantly and continuously enrich our understanding of RNA biology.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Chengqi Yi: 0000-0003-2540-9729 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We thank X. Shu and C. Zhu for their insights and discussions. This work was supported by the National Basic Research Foundation of China (No. MOST2016YFC0900300), the National Natural Science Foundation of China (No. 21522201) and the Beijing Natural Science Foundation (No. 5162012). We apologize for not being able to cite all publications related to this topic due to space constraints of the journal.

■

KEYWORDS RNA modification: natural RNA molecules contain various chemically modified nucleosides, which are derived from the four standard nucleosides (adenosine, guanosine, cytidine, and uridine), and over 100 chemically distinct modifications in RNA have been identified in cellular RNA to date; N6methyladenosine: the most prevalent internal modification in mRNA is methylation at the N6 position of adenosine; 5methylcytosine: the methylation at the C5 position of cytosine is another RNA modification deposited into a wide range of tRNA, rRNA, mRNA, and lncRNA; pseudouridine: known as the “fifth nucleotide” of RNA with the most abundant RNA modification being a C−C glycosidic rotation isomer of uridine; N1-methyladenosine: modification in RNA that has a methyl group in the N1 position of adenosine and is present in tRNA, rRNA, and recently shown in mRNA and lncRNA; 5hydroxymethylcytosine: an oxidation derivative of 5-methylcytosine in RNA catalyzed by ten-eleven translocation (TET) family enzymes; epitranscriptomics: booming and expanding research area of RNA modifications

■

REFERENCES

(1) Song, X., and Nazar, R. N. (2002) Modification of rRNA as a ’quality control mechanism’ in ribosome biogenesis. FEBS Lett. 523, 182−186. (2) Agris, P. F. (2004) Decoding the genome: a modified view. Nucleic Acids Res. 32, 223−238. (3) Karijolich, J., Yi, C., and Yu, Y. T. (2015) Transcriptome-wide dynamics of RNA pseudouridylation. Nat. Rev. Mol. Cell Biol. 16, 581− 585. (4) Mamane, Y., Petroulakis, E., Rong, L., Yoshida, K., Ler, L. W., and Sonenberg, N. (2004) eIF4E–from translation to transformation. Oncogene 23, 3172−3179. (5) Topisirovic, I., Svitkin, Y. V., Sonenberg, N., and Shatkin, A. J. (2011) Cap and cap-binding proteins in the control of gene expression. Wiley Interdiscip. Rev. RNA 2, 277−298. 322

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology (24) Zheng, G., Dahl, J. A., Niu, Y., Fedorcsak, P., Huang, C. M., Li, C. J., Vagbo, C. B., Shi, Y., Wang, W. L., Song, S. H., Lu, Z., Bosmans, R. P., Dai, Q., Hao, Y. J., Yang, X., Zhao, W. M., Tong, W. M., Wang, X. J., Bogdan, F., Furu, K., Fu, Y., Jia, G., Zhao, X., Liu, J., Krokan, H. E., Klungland, A., Yang, Y. G., and He, C. (2013) ALKBH5 Is a Mammalian RNA Demethylase that Impacts RNA Metabolism and Mouse Fertility. Mol. Cell 49, 18−29. (25) Fu, Y., Jia, G., Pang, X., Wang, R. N., Wang, X., Li, C. J., Smemo, S., Dai, Q., Bailey, K. A., Nobrega, M. A., Han, K. L., Cui, Q., and He, C. (2013) FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA. Nat. Commun. 4, 1798. (26) Zheng, G., Dahl, J. A., Niu, Y., Fu, Y., Klungland, A., Yang, Y. G., and He, C. (2013) Sprouts of RNA epigenetics: the discovery of mammalian RNA demethylases. RNA Biol. 10, 915−918. (27) Dunn, D. B. (1961) The occurrence of 1-methyladenine in ribonucleic acid. Biochim. Biophys. Acta 46, 198−200. (28) Hall, R. H. (1963) Method for isolation of 2′-Omethylribonucleosides and N1-methyladenosine from ribonucleic acid. Biochim. Biophys. Acta, Spec. Sect. Nucleic Acids Relat. Subj. 68, 278−283. (29) El Yacoubi, B., Bailly, M., and de Crecy-Lagard, V. (2012) Biosynthesis and function of posttranscriptional modifications of transfer RNAs. Annu. Rev. Genet. 46, 69−95. (30) Sharma, S., Watzinger, P., Kotter, P., and Entian, K. D. (2013) Identification of a novel methyltransferase, Bmt2, responsible for the N-1-methyl-adenosine base modification of 25S rRNA in Saccharomyces cerevisiae. Nucleic Acids Res. 41, 5428−5443. (31) Peifer, C., Sharma, S., Watzinger, P., Lamberth, S., Kotter, P., and Entian, K. D. (2013) Yeast Rrp8p, a novel methyltransferase responsible for m1A 645 base modification of 25S rRNA. Nucleic Acids Res. 41, 1151−1163. (32) Li, X., Xiong, X., Wang, K., Wang, L., Shu, X., Ma, S., and Yi, C. (2016) Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome. Nat. Chem. Biol. 12, 311−316. (33) Dominissini, D., Nachtergaele, S., Moshitch-Moshkovitz, S., Peer, E., Kol, N., Ben-Haim, M. S., Dai, Q., Di Segni, A., SalmonDivon, M., Clark, W. C., Zheng, G., Pan, T., Solomon, O., Eyal, E., Hershkovitz, V., Han, D., Dore, L. C., Amariglio, N., Rechavi, G., and He, C. (2016) The dynamic N(1)-methyladenosine methylome in eukaryotic messenger RNA. Nature 530, 441−446. (34) Helm, M. (2006) Post-transcriptional nucleotide modification and alternative folding of RNA. Nucleic Acids Res. 34, 721−733. (35) Helm, M., Giege, R., and Florentz, C. (1999) A Watson-Crick base-pair-disrupting methyl group (m1A9) is sufficient for cloverleaf folding of human mitochondrial tRNALys. Biochemistry 38, 13338− 13346. (36) Agris, P. F., Sierzputowska-Gracz, H., and Smith, C. (1986) Transfer RNA contains sites of localized positive charge: carbon NMR studies of [13C]methyl-enriched Escherichia coli and yeast tRNAPhe. Biochemistry 25, 5126−5131. (37) Hauenschild, R., Tserovski, L., Schmid, K., Thuring, K., Winz, M. L., Sharma, S., Entian, K. D., Wacheul, L., Lafontaine, D. L., Anderson, J., Alfonzo, J., Hildebrandt, A., Jaschke, A., Motorin, Y., and Helm, M. (2015) The reverse transcription signature of N-1methyladenosine in RNA-Seq is sequence dependent. Nucleic Acids Res. 43, 9950−9964. (38) Waku, T., and Nakajima, Y. (2016) NML-mediated rRNA base methylation links ribosomal subunit formation to cell proliferation in a p53-dependent manner. J. Cell Sci. 129, 2382−2393. (39) Chujo, T., and Suzuki, T. (2012) Trmt61B is a methyltransferase responsible for 1-methyladenosine at position 58 of human mitochondrial tRNAs. RNA 18, 2269−2276. (40) Ozanick, S., Krecic, A., Andersland, J., and Anderson, J. T. (2005) The bipartite structure of the tRNA m1A58 methyltransferase from S. cerevisiae is conserved in humans. RNA 11, 1281−1290. (41) Vilardo, E., Nachbagauer, C., Buzet, A., Taschner, A., Holzmann, J., and Rossmanith, W. (2012) A subcomplex of human mitochondrial RNase P is a bifunctional methyltransferase–extensive moonlighting in mitochondrial tRNA biogenesis. Nucleic Acids Res. 40, 11583−11593.

(42) Liu, F., Clark, W., Luo, G., Wang, X., Fu, Y., Wei, J., Wang, X., Hao, Z., Dai, Q., Zheng, G., Ma, H., Han, D., Evans, M., Klungland, A., Pan, T., and He, C. (2016) ALKBH1-Mediated tRNA Demethylation Regulates Translation. Cell 167, 816−828. (43) Klagsbrun, M. (1973) An evolutionary study of the methylation of transfer and ribosomal ribonucleic acid in prokaryote and eukaryote organisms. J. Biol. Chem. 248, 2612−2620. (44) Motorin, Y., and Helm, M. (2010) tRNA stabilization by modified nucleotides. Biochemistry 49, 4934−4944. (45) Squires, J. E., and Preiss, T. (2010) Function and detection of 5methylcytosine in eukaryotic RNA. Epigenomics 2, 709−715. (46) Chow, C. S., Lamichhane, T. N., and Mahto, S. K. (2007) Expanding the nucleotide repertoire of the ribosome with posttranscriptional modifications. ACS Chem. Biol. 2, 610−619. (47) Squires, J. E., Patel, H. R., Nousch, M., Sibbritt, T., Humphreys, D. T., Parker, B. J., Suter, C. M., and Preiss, T. (2012) Widespread occurrence of 5-methylcytosine in human coding and non-coding RNA. Nucleic Acids Res. 40, 5023−5033. (48) Hussain, S., Sajini, A. A., Blanco, S., Dietmann, S., Lombard, P., Sugimoto, Y., Paramor, M., Gleeson, J. G., Odom, D. T., Ule, J., and Frye, M. (2013) NSun2-mediated cytosine-5 methylation of vault noncoding RNA determines its processing into regulatory small RNAs. Cell Rep. 4, 255−261. (49) Khoddami, V., and Cairns, B. R. (2013) Identification of direct targets and modified bases of RNA cytosine methyltransferases. Nat. Biotechnol. 31, 458−464. (50) Edelheit, S., Schwartz, S., Mumbach, M. R., Wurtzel, O., and Sorek, R. (2013) Transcriptome-wide mapping of 5-methylcytidine RNA modifications in bacteria, archaea, and yeast reveals m5C within archaeal mRNAs. PLoS Genet. 9, e1003602. (51) Fu, L., Guerrero, C. R., Zhong, N., Amato, N. J., Liu, Y., Liu, S., Cai, Q., Ji, D., Jin, S. G., Niedernhofer, L. J., Pfeifer, G. P., Xu, G. L., and Wang, Y. (2014) Tet-mediated formation of 5-hydroxymethylcytosine in RNA. J. Am. Chem. Soc. 136, 11582−11585. (52) Huber, S. M., van Delft, P., Mendil, L., Bachman, M., Smollett, K., Werner, F., Miska, E. A., and Balasubramanian, S. (2015) Formation and abundance of 5-hydroxymethylcytosine in RNA. ChemBioChem 16, 752−755. (53) Delatte, B., Wang, F., Ngoc, L. V., Collignon, E., Bonvin, E., Deplus, R., Calonne, E., Hassabi, B., Putmans, P., Awe, S., Wetzel, C., Kreher, J., Soin, R., Creppe, C., Limbach, P. A., Gueydan, C., Kruys, V., Brehm, A., Minakhina, S., Defrance, M., Steward, R., and Fuks, F. (2016) RNA biochemistry. Transcriptome-wide distribution and function of RNA hydroxymethylcytosine. Science 351, 282−285. (54) Cohn, W. E. (1951) Some results of the applications of ionexchange chromatography to nucleic acid chemistry. J. Cell. Comp. Physiol. 38, 21−40. (55) Cohn, W. E., and Volkin, E. (1951) Nucleoside-5′-Phosphates from Ribonucleic Acid. Nature 167, 483−484. (56) Arnez, J. G., and Steitz, T. A. (1994) Crystal structure of unmodified tRNA(Gln) complexed with glutaminyl-tRNA synthetase and ATP suggests a possible role for pseudo-uridines in stabilization of RNA structure. Biochemistry 33, 7560−7567. (57) Li, X., Ma, S., and Yi, C. (2016) Pseudouridine: the fifth RNA nucleotide with renewed interests. Curr. Opin. Chem. Biol. 33, 108− 116. (58) Schwartz, S., Bernstein, D. A., Mumbach, M. R., Jovanovic, M., Herbst, R. H., Leon-Ricardo, B. X., Engreitz, J. M., Guttman, M., Satija, R., Lander, E. S., Fink, G., and Regev, A. (2014) Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148−162. (59) Carlile, T. M., Rojas-Duran, M. F., Zinshteyn, B., Shin, H., Bartoli, K. M., and Gilbert, W. V. (2014) Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells. Nature 515, 143−146. (60) Lovejoy, A. F., Riordan, D. P., and Brown, P. O. (2014) Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae. PLoS One 9, e110799. 323

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology (61) Li, X., Zhu, P., Ma, S., Song, J., Bai, J., Sun, F., and Yi, C. (2015) Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome. Nat. Chem. Biol. 11, 592−597. (62) Kiss, T., Fayet-Lebaron, E., and Jady, B. E. (2010) Box H/ACA small ribonucleoproteins. Mol. Cell 37, 597−606. (63) Hamma, T., and Ferre-D’Amare, A. R. (2006) Pseudouridine synthases. Chem. Biol. 13, 1125−1135. (64) Spenkuch, F., Motorin, Y., and Helm, M. (2014) Pseudouridine: still mysterious, but never a fake (uridine)! RNA Biol. 11, 1540−1554. (65) Wurm, J. P., Meyer, B., Bahr, U., Held, M., Frolow, O., Kotter, P., Engels, J. W., Heckel, A., Karas, M., Entian, K. D., and Wohnert, J. (2010) The ribosome assembly factor Nep1 responsible for BowenConradi syndrome is a pseudouridine-N1-specific methyltransferase. Nucleic Acids Res. 38, 2387−2398. (66) Alarcon, C. R., Goodarzi, H., Lee, H., Liu, X., Tavazoie, S., and Tavazoie, S. F. (2015) HNRNPA2B1 Is a Mediator of m(6)ADependent Nuclear RNA Processing Events. Cell 162, 1299−1308. (67) Alarcon, C. R., Lee, H., Goodarzi, H., Halberg, N., and Tavazoie, S. F. (2015) N6-methyladenosine marks primary microRNAs for processing. Nature 519, 482−485. (68) Liu, N., Dai, Q., Zheng, G., He, C., Parisien, M., and Pan, T. (2015) N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature 518, 560−564. (69) Xiao, W., Adhikari, S., Dahal, U., Chen, Y. S., Hao, Y. J., Sun, B. F., Sun, H. Y., Li, A., Ping, X. L., Lai, W. Y., Wang, X., Ma, H. L., Huang, C. M., Yang, Y., Huang, N., Jiang, G. B., Wang, H. L., Zhou, Q., Wang, X. J., Zhao, Y. L., and Yang, Y. G. (2016) Nuclear m(6)A Reader YTHDC1 Regulates mRNA Splicing. Mol. Cell 61, 507−519. (70) Zhao, X., Yang, Y., Sun, B. F., Shi, Y., Yang, X., Xiao, W., Hao, Y. J., Ping, X. L., Chen, Y. S., Wang, W. J., Jin, K. X., Wang, X., Huang, C. M., Fu, Y., Ge, X. M., Song, S. H., Jeong, H. S., Yanagisawa, H., Niu, Y., Jia, G. F., Wu, W., Tong, W. M., Okamoto, A., He, C., Rendtlew Danielsen, J. M., Wang, X. J., and Yang, Y. G. (2014) FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 24, 1403−1419. (71) Molinie, B., Wang, J., Lim, K. S., Hillebrand, R., and Lu, Z. X. (2016) m(6)A-LAIC-seq reveals the census and complexity of the m(6)A epitranscriptome. Nat. Methods 13, 692−698. (72) Ke, S., Alemu, E. A., Mertens, C., Gantman, E. C., Fak, J. J., Mele, A., Haripal, B., Zucker-Scharff, I., Moore, M. J., Park, C. Y., Vagbo, C. B., Kussnierczyk, A., Klungland, A., Darnell, J. E., Jr., and Darnell, R. B. (2015) A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation. Genes Dev. 29, 2037− 2053. (73) Wang, X., Lu, Z., Gomez, A., Hon, G. C., Yue, Y., Han, D., Fu, Y., Parisien, M., Dai, Q., Jia, G., Ren, B., Pan, T., and He, C. (2014) N6-methyladenosine-dependent regulation of messenger RNA stability. Nature 505, 117−120. (74) Geula, S., Moshitch-Moshkovitz, S., Dominissini, D., Mansour, A. A., Kol, N., Salmon-Divon, M., Hershkovitz, V., Peer, E., Mor, N., Manor, Y. S., Ben-Haim, M. S., Eyal, E., Yunger, S., Pinto, Y., Jaitin, D. A., Viukov, S., Rais, Y., Krupalnik, V., Chomsky, E., Zerbib, M., Maza, I., Rechavi, Y., Massarwa, R., Hanna, S., Amit, I., Levanon, E. Y., Amariglio, N., Stern-Ginossar, N., Novershtern, N., Rechavi, G., and Hanna, J. H. (2015) Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation. Science 347, 1002−1006. (75) Wang, X., Zhao, B. S., Roundtree, I. A., Lu, Z., Han, D., Ma, H., Weng, X., Chen, K., Shi, H., and He, C. (2015) N(6)-methyladenosine Modulates Messenger RNA Translation Efficiency. Cell 161, 1388− 1399. (76) Meyer, K. D., Patil, D. P., Zhou, J., Zinoviev, A., Skabkin, M. A., Elemento, O., Pestova, T. V., Qian, S. B., and Jaffrey, S. R. (2015) 5′ UTR m(6)A Promotes Cap-Independent Translation. Cell 163, 999− 1010. (77) Zhou, J., Wan, J., Gao, X., Zhang, X., Jaffrey, S. R., and Qian, S. B. (2015) Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature 526, 591−594.

(78) Lin, S., Choe, J., Du, P., Triboulet, R., and Gregory, R. I. (2016) The m(6)A Methyltransferase METTL3 Promotes Translation in Human Cancer Cells. Mol. Cell 62, 335−345. (79) Zhao, B. S., and He, C. (2015) Fate by RNA methylation: m6A steers stem cell pluripotency. Genome Biol. 16, 43. (80) Chen, T., Hao, Y. J., Zhang, Y., Li, M. M., Wang, M., Han, W., Wu, Y., Lv, Y., Hao, J., Wang, L., Li, A., Yang, Y., Jin, K. X., Zhao, X., Li, Y., Ping, X. L., Lai, W. Y., Wu, L. G., Jiang, G., Wang, H. L., Sang, L., Wang, X. J., Yang, Y. G., and Zhou, Q. (2015) m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency. Cell Stem Cell 16, 289−301. (81) Batista, P. J., Molinie, B., Wang, J., Qu, K., Zhang, J., Li, L., Bouley, D. M., Lujan, E., Haddad, B., Daneshvar, K., Carter, A. C., Flynn, R. A., Zhou, C., Lim, K. S., Dedon, P., Wernig, M., Mullen, A. C., Xing, Y., Giallourakis, C. C., and Chang, H. Y. (2014) m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells. Cell Stem Cell 15, 707−719. (82) Aguilo, F., Zhang, F., Sancho, A., Fidalgo, M., Di Cecilia, S., Vashisht, A., Lee, D. F., Chen, C. H., Rengasamy, M., Andino, B., Jahouh, F., Roman, A., Krig, S. R., Wang, R., Zhang, W., Wohlschlegel, J. A., Wang, J., and Walsh, M. J. (2015) Coordination of m(6)A mRNA Methylation and Gene Transcription by ZFP217 Regulates Pluripotency and Reprogramming. Cell Stem Cell 17, 689−704. (83) Patil, D. P., Chen, C. K., Pickering, B. F., Chow, A., Jackson, C., Guttman, M., and Jaffrey, S. R. (2016) m6A RNA methylation promotes XIST-mediated transcriptional repression. Nature 537, 369− 373. (84) Lichinchi, G., Gao, S., Saletore, Y., and Gonzalez, G. M. (2016) Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells. Nat. Microbiol. 1, 16011. (85) Kennedy, E. M., Bogerd, H. P., Kornepati, A. V., Kang, D., Ghoshal, D., Marshall, J. B., Poling, B. C., Tsai, K., Gokhale, N. S., Horner, S. M., and Cullen, B. R. (2016) Posttranscriptional m(6)A Editing of HIV-1 mRNAs Enhances Viral Gene Expression. Cell Host Microbe 19, 675−685. (86) Tirumuru, N., Zhao, B. S., Lu, W., Lu, Z., He, C., and Wu, L. (2016) N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression. eLife 5, e15528. (87) Lichinchi, G., Zhao, B. S., Wu, Y., Lu, Z., Qin, Y., He, C., and Rana, T. M. (2016) Dynamics of Human and Viral RNA Methylation during Zika Virus Infection. Cell Host Microbe 20, 666−673. (88) Gokhale, N. S., McIntyre, A. B., McFadden, M. J., Roder, A. E., Kennedy, E. M., Gandara, J. A., Hopcraft, S. E., Quicke, K. M., Vazquez, C., Willer, J., Ilkayeva, O. R., Law, B. A., Holley, C. L., GarciaBlanco, M. A., Evans, M. J., Suthar, M. S., Bradrick, S. S., Mason, C. E., and Horner, S. M. (2016) N6-Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection. Cell Host Microbe 20, 654−665. (89) Karikó, K., Buckstein, M., Ni, H., and Weissman, D. (2005) Suppression of RNA Recognition by Toll-like Receptors: The Impact of Nucleoside Modification and the Evolutionary Origin of RNA. Immunity 23, 165−175. (90) Hull, C. M., and Bevilacqua, P. C. (2016) Discriminating Self and Non-Self by RNA: Roles for RNA Structure, Misfolding, and Modification in Regulating the Innate Immune Sensor PKR. Acc. Chem. Res. 49, 1242−1249. (91) Kariko, K., Muramatsu, H., Ludwig, J., and Weissman, D. (2011) Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA. Nucleic Acids Res. 39, e142. (92) Kariko, K., Muramatsu, H., Keller, J. M., and Weissman, D. (2012) Increased erythropoiesis in mice injected with submicrogram quantities of pseudouridine-containing mRNA encoding erythropoietin. Mol. Ther. 20, 948−953. (93) Chen, C., Zhao, X., Kierzek, R., and Yu, Y. T. (2010) A flexible RNA backbone within the polypyrimidine tract is required for U2AF65 binding and pre-mRNA splicing in vivo. Mol. Cell. Biol. 30, 4108−4119. (94) Kariko, K., Muramatsu, H., Welsh, F. A., Ludwig, J., Kato, H., Akira, S., and Weissman, D. (2008) Incorporation of pseudouridine 324

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325

Reviews

ACS Chemical Biology into mRNA yields superior nonimmunogenic vector with increased translational capacity and biological stability. Mol. Ther. 16, 1833− 1840. (95) Hoernes, T. P., Clementi, N., Faserl, K., Glasner, H., Breuker, K., Lindner, H., Huttenhofer, A., and Erlacher, M. D. (2016) Nucleotide modifications within bacterial messenger RNAs regulate their translation and are able to rewire the genetic code. Nucleic Acids Res. 44, 852−862. (96) Karijolich, J., and Yu, Y. T. (2011) Converting nonsense codons into sense codons by targeted pseudouridylation. Nature 474, 395− 398. (97) Fernandez, I. S., Ng, C. L., Kelley, A. C., Wu, G., Yu, Y. T., and Ramakrishnan, V. (2013) Unusual base pairing during the decoding of a stop codon by the ribosome. Nature 500, 107−110. (98) Hoernes, T. P., and Erlacher, M. D. (2017) Translating the epitranscriptome. Wiley Interdiscip. Rev. RNA 8, e1375. (99) Dominissini, D., Moshitch-Moshkovitz, S., Schwartz, S., SalmonDivon, M., Ungar, L., Osenberg, S., Cesarkas, K., Jacob-Hirsch, J., Amariglio, N., Kupiec, M., Sorek, R., and Rechavi, G. (2012) Topology of the human and mouse m6A RNA methylomes revealed by m6Aseq. Nature 485, 201−206. (100) Meyer, K. D., Saletore, Y., Zumbo, P., Elemento, O., Mason, C. E., and Jaffrey, S. R. (2012) Comprehensive analysis of mRNA methylation reveals enrichment in 3′ UTRs and near stop codons. Cell 149, 1635−1646. (101) Schwartz, S., Agarwala, S. D., Mumbach, M. R., Jovanovic, M., Mertins, P., Shishkin, A., Tabach, Y., Mikkelsen, T. S., Satija, R., Ruvkun, G., Carr, S. A., Lander, E. S., Fink, G. R., and Regev, A. (2013) High-resolution mapping reveals a conserved, widespread, dynamic mRNA methylation program in yeast meiosis. Cell 155, 1409−1421. (102) Luo, G. Z., MacQueen, A., Zheng, G., Duan, H., Dore, L. C., Lu, Z., Liu, J., Chen, K., Jia, G., Bergelson, J., and He, C. (2014) Unique features of the m6A methylome in Arabidopsis thaliana. Nat. Commun. 5, 5630. (103) Linder, B., Grozhik, A. V., Olarerin-George, A. O., Meydan, C., Mason, C. E., and Jaffrey, S. R. (2015) Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods 12, 767−772. (104) Chen, K., Lu, Z., Wang, X., Fu, Y., Luo, G. Z., Liu, N., Han, D., Dominissini, D., Dai, Q., Pan, T., and He, C. (2015) High-resolution N(6) -methyladenosine (m(6) A) map using photo-crosslinkingassisted m(6) A sequencing. Angew. Chem., Int. Ed. 54, 1587−1590. (105) Macon, J. B., and Wolfenden, R. (1968) 1-Methyladenosine. Dimroth rearrangement and reversible reduction. Biochemistry 7, 3453−3458. (106) Schaefer, M., Pollex, T., Hanna, K., and Lyko, F. (2009) RNA cytosine methylation analysis by bisulfite sequencing. Nucleic Acids Res. 37, e12. (107) Gilham, P. T. (1962) An Addition Reaction Specific for Uridine and Guanosine Nucleotides and its Application to the Modification of Ribonuclease Action. J. Am. Chem. Soc. 84, 687−688. (108) Bakin, A., and Ofengand, J. (1993) Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique. Biochemistry 32, 9754−9762. (109) Li, X., Ma, S., and Yi, C. (2015) Pseudouridine Chemical Labeling and Profiling. Methods Enzymol. 560, 247−272. (110) Gilbert, W. V., Bell, T. A., and Schaening, C. (2016) Messenger RNA modifications: Form, distribution, and function. Science 352, 1408−1412. (111) Hussain, S., Aleksic, J., Blanco, S., Dietmann, S., and Frye, M. (2013) Characterizing 5-methylcytosine in the mammalian epitranscriptome. Genome Biol. 14, 215. (112) Shafik, A., Schumann, U., Evers, M., Sibbritt, T., and Preiss, T. (2016) The emerging epitranscriptomics of long noncoding RNAs. Biochim. Biophys. Acta, Gene Regul. Mech. 1859, 59−70.

325

DOI: 10.1021/acschembio.6b00960 ACS Chem. Biol. 2017, 12, 316−325