Chemical Modifications to RNA: A New Layer of Gene Expression

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Review

Chemical Modifications to RNA: a New Layer of Gene Expression Regulation Jinghui Song, and Chengqi Yi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00960 • Publication Date (Web): 04 Jan 2017 Downloaded from http://pubs.acs.org on January 5, 2017

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ACS Chemical Biology

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, Peking University, Beijing 100871, China. ‡

Department of Chemical Biology and Synthetic and Functional Biomolecules Center,

College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.

ACS Paragon Plus Environment


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ABSTRACT The first chemical modification to RNA was discovered nearly 60 years ago; up to now, 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.


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Besides 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 non-coding RNAs (ncRNA), such as ribosomal RNA (rRNA), transfer RNA (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 non-coding 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),

5-hydroxymethylcytosine (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. Since 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 enzymes, high-throughput detection methods, and



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biological functions.

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: while the syn conformation is more energetically favored than the anti conformation in single-stranded RNA (ssRNA),11, 12 the methyl group in m6A must adopt to a high-energy anti orientation in paired RNA, in order to accommodate normal Waston-Crick base-pair.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 1-associating protein (WTAP).15-19 In the complex, METTL3 serves as the catalytically active subunit, METTL14 enhances substrate recognition and RNA binding, while WTAP may facilitate the translocation into nuclear speckles.19-21 Biochemical analyses have revealed that the consensus substrate 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


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intermediates N6-hydroxymethyladenosine (hm6A) and N6-formyladenosine (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 to the N1 position of adenosine, forming m1A (Figure 1). m1A was first discovered in the 1960s,27, 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, 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 negative charged phosphates in the backbone.34-36 Furthermore, due to the methyl group in the N1 position of m1A at the Watson-Crick interface, m1A can interfere 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



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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 RNA structure.56 Ψ is the most abundant RNA modification, presented 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 RNA-dependent pseudouridine synthases (PUS) that require the cofactor, box H/ACA ribonucleoproteins, as guider to recognize different substrates; another is catalyzed by the RNA-independent PUS that require no cofactor.57, 62, 63 10 PUSs were identified in yeast: Cbf5 is a RNA-dependent PUS; the rest Pus1-9 are RNA-independent.64 13 PUSs were identified in human: only DKC1 (a homolog of Cbf5) is a RNA-dependent


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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 about 0.4%-0.7%.16, 23, 24

The ratio of Ψ/U in mammalian mRNA is about 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 about 0.02-0.09%; the hm5C/C ratio is about 0.001%-0.003%, only 1/25 of m5C.52 The m1A/A ratio in mammalian mRNA is about 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.

BIOLOGICAL FUNCTIONS With the development of high-throughput technologies of m6A, more and more functions of m6A were 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 evidences for the regulatory role of m6A in pre-mRNA and pri-miRNA processing (Figure 2). For pri-miRNA processing, m6A is



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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 exon through recruiting SRSF3 while blocking the binding of SRSF10 to mRNA.69 The increased m6A level could also promote 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 nucleus, it is crucial that mature mRNA are exported to the cytoplasm. Researchers found that nuclear mRNA export is accelerated upon ALKBH5 knockdown, indicating that m6A might involve in mRNA export (Figure 2).24 After exporting 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 mRNA via the translocation of bound mRNA from the translatable pool to processing bodies.73 These results revealed the tight regulations between m 6A modification and mRNA decay (Figure 2). On the other hand, the m6A mark can also affect the translation


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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 XIST-mediated 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



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RNA. Moreover, one study found that binding of YTHDF1–3 proteins to m6A-methylated HIV-1 RNA promotes viral protein and RNA expression,85 while another recent study found that YTHDF1–3 proteins bind m6A-modified 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 Flaviviridae virus.87, 88 These data suggested the important role of m6A in viral infection. Given that cell can discriminate self and non-self 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 3’ splice site) of artificial pre-mRNA inhibits pre-mRNA splicing by affecting U2 auxiliary factor (U2AF) binding in Xenopus oocytes.93 Yet, 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, Pus7-dependent 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 complicated manners. When Ψ-containing 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 to translation were also


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observed. One study has reported that the incorporation of a single Ψ at each position of the codon ‘UUU’ of bacterial 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 converting 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 m6A 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% amounts of translation product using bacterial whole cell extract; a m5C at the second nucleotide of the codon ‘CCC’ could induce recoding.95 In addition, uncapped mRNAs that are randomly incorporated by 50% m5C significantly decrease translation in HeLa cell extracts.76 In contrast to m5C which negatively impact 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 correlates 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 a resolution of 100-200 nt were generated in 2012 by two independent groups based on m6A-specific antibody



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(m6A/MeRIP-seq).99, 100 Due to the lack of chemical methods to differentiate m6A from A, these approaches used an m6A-specifc antibody to pull down m6A-containing RNA (but not non-m6A RNA), which allows pre-enrichment of m6A-containing RNA for high-throughput sequencing; they were quickly adopted by many other labs and used frequently today. Such methods identified thousands of m6A peaks in mammalian mRNA and lncRNA: the m6A peaks are enriched in 3’UTR and near stop codons. Besides, m6A is also a widespread modification in other species mRNA: in yeast, m6A is dynamically regulated methylation during meiosis;101 in plant, m6A peaks are abundant around the start codon (16%).102 In order to reach single-base-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 crosslinks between antibody and m6A-containing RNA.72, 103 During RT, the antibody-RNA crosslinking sites can cause the mutational or truncational signatures of cDNA, so base-resolution m6A sites can be identified. In PA-m6A-seq, photoreactive nucleoside analog (4-thiouridine, 4SU) that can improve the crosslinking efficiency was added to the growth medium, and then incorporated into newly synthesized RNA molecules; after immunoprecipitation, covalent crosslinks 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 crosslinking sites, so it is possible to identify m6A at base-resolution. Recently, a new modified strategy, called m6A-LAIC-seq,

was

developed

to

quantify

the

m 6A


stoichiometry

on

a

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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. 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 m1A-ID-seq and m1A-seq, respectively) 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 confident m1A peaks can be identified. Specially, m1A-seq exploited Dimroth rearrangement to convert m1A to m6A under alkaline conditions, since 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 firstly 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 m5dC 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 high-throughput sequencing, indicating the presence of m5C



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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 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 transcriptome-wide targets of NSUN2. In m5C-RIP approach, an antibody against m5C was used to validate m5C sites in archaea mRNA, but m5C-RIP has not been applied to mammalian cells as yet. Such antibody-based approach could in principle be applied to other modifications as well. For instance, a method utilizing a 5hmC-antibody (“called hMeRIP-seq”) was reported to identify more than 3,000 hm5C peaks in 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 blot. The transcriptome-wide mapping of Ψ sites is relied on a 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), while 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


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(Figure 5a). Three independent CMC-based profiling methods of Ψ (called Ψ-Seq, Pseudo-Seq, and PSI-Seq, respectively) have identified about 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 CeU-Seq, a CMC derivative, azido-CMC (N3-CMC) was used to label Ψ and the N3-CMC-Ψ adduct can be coupled with biotin via ‘click’ chemistry109. 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.

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 signal to noise ratio. Another 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. The major concerns to detect RNA modifications are the precision, sensitivity, quantitative ability, and resolution of high-throughput detection methods. However, many challenges remain to be solved.



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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 unbiased dissect the

distribution of m6A. 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 depth 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 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, over-expression of methyltransferase are 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 are needed to develop in 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 is future Ψ profiling are needed, e.g., finding new chemicals that specifically react with Ψ. For hm5C and m1A detection, current sequencing


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technologies have not reached single-base-resolution, which hinders the functional studies of RNA modifications. Learning from the success of single-base and quantitative m6A sequencing technologies, optimized sequencing methods of hm5C and m1A are expected in future. Other considerations are also required for functional studies of RNA modifications. Firstly, 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-LAIC-seq, most sequencing technologies cannot quantify the percentage of transcripts containing modification. Secondly, it is still unknown whether any correlations exist among different RNA modifications. Multiple RNA modifications may contribute together to regulate the biological consequences for a certain transcript. Therefore, it will be helpful to develop high-throughput technologies that can identify multiple modifications at a same molecule simultaneously. Thirdly, all existing RNA modifications sequencing methods cannot be applied to single-cell directly. It is known that m6A has the 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, the sequencing technologies of RNA modifications are just beginning. We envision that future tools, particularly novel chemical biology methods, could be applied to further facilitate the studies in the field of epitranscriptomics.



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CONCLUDING REMARKS Great progress on RNA modifications has been achieved during the past 5 years due to the rapid development of novel RNA modifications 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 evidences also suggest 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. Firstly, 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 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 the vital input from the chemical biology field will significantly and continuously enrich our understanding of RNA biology.

AUTHOR INFORMATION


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Corresponding Author *

E-mail: [email protected]

Notes The authors declare that they have no conflict of 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 Beijing Natural Science Foundation (no. 5162012). We apologize for not being able to cite all the publications related to this topic due to space constrain 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). Over 100 chemically distinct modifications in RNA have been

identified in cellular RNA so far. N6-methyladenosine: The most prevalent internal modification in messenger RNA is methylation at the N6 position of adenosine. 5-methylcytosine: The methylation at the C5 position of cytosine is another RNA modification, deposited into a wide range of tRNA, rRNA, mRNA, and lncRNA.



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Pseudouridine: Known as the ‘fifth nucleotide’ of RNA, the most abundant RNA modification is a C-C glycosidic rotation isomer of uridine. N1-methyladenosine: A modification in RNA, which has a methyl group in the N1 of position of adenosine and is present in tRNA, rRNA, and recently shown in mRNA and lncRNA. 5-hydroxymethylcytosine: 5-hydroxymethylcytosine is an oxidation derivative of 5-methylcytosine in RNA, catalyzed by ten-eleven translocation (TET) family enzymes. Epitranscriptomics: Epitranscriptomics is a booming and expending research area of RNA modifications.

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Figure legends 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 nucleus, RNA modifications affect pre-mRNA and pri-miRNA


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processing, and nuclear export. In cytoplasm, RNA modifications regulates mRNA translation and stability. Red and black lines represent RNA and DNA, respectively. RNA modification is indicated by blue filled circle.

Figure 3. Dimroth rearrangement and demethylase can eliminate the RT signatures of m1A. a) m1A in RNA causes stop or misincorporation during RT, while 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.

Figure 4. Bisulfite-mediated conversion of cytosine to uracil. a) the principle of RNA bisulfite sequencing; b) cytosine deamination via bisulfite treatment.

Figure 5. CMC specifically labels Ψ. a) the work flow of CMC labeling; b) chemical structure of CMC; c) CMC can acylate U, Ψ, and G; d) after alkaline treatment, only the adducts of CMC-Ψ are remained.



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Graphical Table of Contents 69x40mm (300 x 300 DPI)


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Figure 1. Chemical structures of m6A, m1A, m5C, hm5C, and Ψ. 140x21mm (300 x 300 DPI)



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Figure 2. Dynamic RNA modifications in mRNA and lncRNA affect a wide range of cellular processes. In nucleus, RNA modifications affect pre-mRNA and pri-miRNA processing, and nuclear export. In cytoplasm, RNA modifications regulates mRNA translation and stability. Red and black lines represent RNA and DNA, respectively. RNA modification is indicated by blue filled circle. 140x84mm (300 x 300 DPI)


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Figure 3. Dimroth rearrangement and demethylase can eliminate the RT signatures of m1A. a) m1A in RNA causes stop or misincorporation during RT, while 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. 106x97mm (300 x 300 DPI)



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Figure 4. Bisulfite-mediated conversion of cytosine to uracil. a) the principle of RNA bisulfite sequencing; b) cytosine deamination via bisulfite treatment. 107x54mm (300 x 300 DPI)


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Figure 5. CMC specifically labels Ψ. a) the work flow of CMC labeling; b) chemical structure of CMC; c) CMC can acylate U, Ψ, and G; d) after alkaline treatment, only the adducts of CMC-Ψ are remained. 110x92mm (300 x 300 DPI)


Chemical Modifications to RNA: A New Layer of Gene Expression

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