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Analytical Methods for Deciphering RNA Modifications Bei Chen, Bi-Feng Yuan, and Yu-Qi Feng Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04078 • Publication Date (Web): 13 Sep 2018 Downloaded from http://pubs.acs.org on September 14, 2018

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Analytical Chemistry

Analytical Methods for Deciphering RNA Modifications

Bei Chen, Bi-Feng Yuan,* Yu-Qi Feng

Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Department of Chemistry, Wuhan University, Wuhan 430072, P.R. China Tel.:+86-27-68755595; fax: +86-27-68755595. E-mail address: [email protected]

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1 Introduction RNA molecules harbor various levels of diverse chemical modifications.1 So far, more than 150 structurally distinct modifications have been identified in cellular RNAs.2 Different with previous views that these modifications are mostly static, recent advances demonstrate a much more dynamic landscape of RNA modifications.3 The diverse and dynamic modifications do not affect RNA sequence, but they exert essential and critical influences in a variety of cellular processes in eukaryotic organisms.4 In addition to maintaining structure and catalytic activity, RNA modifications have been considered to serve as additional layer of information carrier on regulating cell physiology, indicating a previously invisible code that is outside their sequences.5,6 Covalent chemical modifications occur in almost all types of RNAs. Transfer RNA (tRNA) contains the richest source of modifications with up to 25% of its nucleotides being modified.7 Ribosomal RNA (rRNA) is the most abundant RNA and also provides numerous modification types located in different sequence and structural contexts.8 In addition to the high diversity and large numbers of modifications in these abundant non-coding RNA, an increasing number of modifications were also discovered in less-abundant species, including messenger RNA (mRNA),9 small nuclear RNA (snRNA),10 and microRNA (miRNA).11,12 However, many exciting functions of RNA modifications remain to be explored. The elucidation of the diverse biological functions of RNA modifications relies the accurate detection, quantification and mapping of these modifications.13 RNA modifications are typically of low abundance and frequently are not detectable by standard detection methods of molecular biology. Methods for deciphering RNA modifications have greatly 2


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improved over the last decade.13 Early detection of RNA modifications mainly relied on liquid chromatography,14 reverse transcriptase - polymerase chain reaction (RT-PCR),15 thin layer chromatography (TLC),16 and capillary electrophoresis

17

for structural elucidation of

modifications, but soon mass spectrometry,18 chemical labeling,19 and next-generation sequencing techniques

20

have been frequently employed to RNA modifications study. A

combination of technical progress and advancement of science even achieved the sensitive analysis of RNA modifications in the single molecule level.21 More recent progress in high-throughput techniques, including next-generation sequencing, allow genome-wide mapping for a variety of chemically distinct RNA modifications, which greatly revolutionize the field of RNA modifications.22 In this review, we summarize and discuss the established technology as well as breakthrough of the techniques mainly in the recent five years (2014-2018) for study of RNA modifications. Importantly, high-throughput detection techniques witness an ever-increasing number of RNA modifications study.23 However, because the in-depth investigation of RNA modifications occurs only recently, just a few research groups have developed relevant expertise for effective detection and mapping of RNA modifications. To this end, we discuss the principles, advantages and weaknesses of these established methods. We believe that with methods development, researchers will continue to uncover functional consequences of known RNA modifications. The improvement of analytical methods will certainly promote the elucidation of RNA modifications, which can provide new opportunities for researchers to manipulate the modification status, and develop small molecules to alter the RNA functions for fundamental research and therapeutic purposes in future. 3



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2 Overall detection of RNA modifications Based on the research purpose, we categorize the methods for analysis of RNA modifications mainly in two groups, overall detection (Figure 1) and location analysis (Figure 2). As for the overall detection, RNA samples are routinely digested to nucleosides or nucleotides and then analyzed by various methods, including capillary electrophoresis (CE), liquid chromatography (LC), liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), fluorescence labeling and chemical labeling-based analysis (Figure 1).

2.1 Capillary electrophoresis The principle of capillary electrophoresis (CE) is based on the separation of charged particles in an electrical field. CE can offer high resolution and fast separation of analytes.24 Several detection techniques can be coupled to CE, including UV-absorbance, MS and laser-induced florescence (LIF) detection. Cornelius et al. on

the

17

separation

developed a sensitive method for analysis of RNA components based of

the

nucleoside-5’-monophosphates

4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl

conjugated ethylene

with diamine

hydrochloride (BODIPY FL EDA) using CE-LIF. After digestion of RNA to nucleoside-5’-monophosphates by nuclease P1, the fluorescence BODIPY conjugates were detected and resolved by CE-LIF without further purification steps. With this method, modified nucleosides, including inosine (I), xanthosine (X), pseudouridine (Ψ) and 4


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2’-O-methyladenosine (Am) from RNA of Drosophila, human liver, human kidney and S. cerevisiae were identified with the limits of detection (LODs) being in the range of 80-200 pM.

This

study

demonstrated

good

potential

of

BODIPY

conjugated

nucleoside-5’-monophosphates detected by CE-LIF to determine RNA composition. Recently, Stephen et al.

25

used CE-LIF to detect adenosine (A) and inosine (I). In this

study, A and I were first labeled with 2,4,6-trinitrobenzenesulfonic to yield fluorescent trinitrophenylated complexes of TNP-A and TNP-I. The TNP-A and TNP-I can further form complexes with γ-cyclodextrin, leading to ~ 25-fold fluorescence enhancement. A and I were simultaneously analyzed and quantified in homogenized rat forebrain samples in less than 10 min with the LODs being 1.6 µM and 4 µM, respectively. However, the separation reproducibility of CE can be affected by many factors and the sample loading volume is normally limited, which requires further improvement.

2.2 Liquid chromatography Liquid chromatography (LC) techniques are well established and commonly used in the analysis of nucleosides and nucleotides with photometric and electrochemical detection. As for the LC-based methods, the baseline separation is critical for the detection since the analysis mainly relies on the chromatographic separation. Anion exchange resins are frequently used in early studies of nucleosides. Desrosiers et al.

26

investigated the methylation in mRNA from Novikoff hepatoma cells using

diethylaminoethyl cellulose DEAE-cellulose chromatography. The composition of the modified nucleosides in RNA was analyzed after enzymatic digestion to nucleosides. By 5



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comparing with the standards, five RNA modifications, including N6-methyladenine (m6A), Am, 2’-O-methylguanosine (Gm), 2’-O-methylcytidine (Cm) and 2’-O-methyluridine (Um), were identified in mRNA. In addition, 29 modifications in tRNA from S. typhimurium and E. coli were also reported by LC-UV analysis.14 Later, Gehrke et al.

27

performed a

comprehensive study by LC-UV method to separate 65 nucleosides in a single run with identification by comparing the retention times to standards. This protocol allowed to determine 31 different nucleosides from 1 µg of tRNA. Zhao et al.

28

further compared the analysis of RNA modifications by HPLC and UPLC.

As a result, 79 peaks were found by HPLC-UV detection and 92 by UPLC-UV detection, suggesting UPLC yielded more peaks of high-performance efficiency. However, due to the weak qualitative capability of the LC-based detection, LC coupled with MS analysis that exhibits much powerful detection performance in terms of the detection sensitivity and identification capability is prevailed in the overall detection of RNA modifications.

2.3 Mass spectrometry As nearly all of these modifications result in an increase in the mass of canonical nucleosides, MS has long been a powerful approach for identifying and characterizing RNA modifications.29 As shown over 20 years ago by McCloskey et al., MS-based methods provide direct identification on modifications that change the molecular weights of canonical nucleosides.30 Recently, significant advances have been made in method development and software for interpreting tandem mass spectra that can provide qualitative and quantitative information on RNA modifications.18 Now the MS-based analysis can enable detection in the 6


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femtomol to attomol levels of RNA modifications. The chromatographic retention times, m/z and fragmentation patterns generated by MS-based analysis provide the identification information of modifications in RNA. Nearly all the reported RNA modifications collected in the databases of “Modomics”

31

and “The RNA

Modification Database”,32 were initially discovered or characterized by MS. MS has been demonstrated to be one of the most powerful tools for the discovery and confirmation of new RNA modifications.

2.3.1 Liquid chromatography-mass spectrometry Liquid chromatography-mass spectrometry (LC-MS) is probably the most widely used MS-based detection platform for characterizing and quantifying RNA modifications.33-35 LC-MS has been used in multiple settings for investigating modifications in specific RNA at the nucleoside level.36,37 RNA sample is routinely digested to nucleosides and then analyzed by LC-MS (Figure 3A). The confirmation of modified nucleosides is typically based on comparison to synthetic standards or by RNA modification databases. Dedon’s group developed a highly precise LC-MS method with multiple reaction monitoring (MRM) mode to quantify changes in the spectrum of tRNA modifications in yeast exposed to toxicants.38,39 [15N5]-2’-deoxyadenosine was used as an internal standard for the quantification of modified nucleosides. The results suggested that 23 of the 25 known tRNA modifications in yeast were identified. The relative proportions of modifications can be quantified in several micrograms of tRNA in a 15-min LC-MS run. Isotope standards are also frequently used for identification and quantification in 7



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MS-based detection.40,41 Wang’s group employed isotope dilution LC-MS with selected ion monitoring (SIM) of protonated molecular ions to quantitatively measure hm5C in RNA.42 RNA samples were enzymatically digested to nucleosides, then [1,3-15N2]hm5C were added as internal standards. Similarly strategy was also employed in the detection and quantification of 5-methylcytidine (m5C), Cm, Am, and m6A in RNA from mammalian cells and tissues.43 The use of an isotope internal standard provides more accurate measurements by compensating for variations due to matrix effects and sample handling. Our group recently utilized LC-ESI-MS/MS to quantitatively analyze DNA and RNA methylation in a single cell.44 We developed a combined strategy of sample preparation for cell lysis, nucleic acids digestion, and nucleosides extraction followed by LC-ESI-MS/MS analysis.

Using

this

strategy,

we

achieved

the

detection

of

m5C,

m6A,

and

5-methyl-2’-deoxycytidine (m5dC) in a single cell. In addition, using an effective circulating tumor cells (CTCs) capture system, we realized the analysis of these modifications in human CTCs and demonstrated the DNA hypomethylation and RNA hypermethylation in CTCs. Positional isomers of nucleoside modifications are difficult to be differentiated in LC-MS analysis due to their similar chromatographic behavior and CID mass spectra. To address this challenge, Jora et al. 45 developed a higher-energy collisional dissociation (HCD) fragmentation technique to generate nucleoside-specific product ion fingerprints for the identification of positional isomers. By increasing the collision energy to 80, the isomers m3C, m4C and m5C were distinctly differentiated by their unique product ion spectrum. Chan et al.

33

employed LC-MS to define the spectra of modifications in tRNA from

Mycobacterium bovis BCG. Enzymatically digested tRNA was analyzed by LC-MS with 8


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neutral loss as well as MRM detection modes. The results indicated 12 modifications were identified by comparing with synthetic standards and 5 tentative modifications by searching the RNA modification databases. Interestingly, N6,N6-dimethyladenosine (m62A) was firstly discovered and characterized in Mycobacterium bovis BCG tRNA. Similarly, Suzuki’s group identified

5-methoxycarbonylmethyl-2-thiouridine

(mcm5s2U)

and

5-methoxycarbonylmethyl-2’-O-methyluridine (mcm5Um) in Leishmania tarentolae tRNA,46 N1-methylgunanosine (m1G) and wybutosine (yW) in S. cerevisiae tRNAPhe by LC-MS analysis.47 MS-based detection often requires synthetic standards; however, many RNA modification standards are not commercially available. To address this issue, Delft et al.

48

recently developed a metabolic labelling approach to achieve generation of stable isotope labeled (SIL) nucleosides, which were then used as reference standards to characterize the RNA modifications by untargeted LC high-resolution mass spectrometry (LC-HRMS) approach. In this strategy, C. elegans larvae were fed with heavy-labeled E. coli that were grown in media containing D-[13C6]glucose and amino acids, or with unlabeled E. coli. Total RNA from labelled or unlabeled C. elegans was isolated and subjected to LC-HRMS analysis. With this method, they identified 21 and 26 modifications in the C. elegans large- and small-RNA

fractions,

respectively.

Interestingly,

they

found

the

changes

of

5-methoxycarbonylmethyl-2-thiouridine (mcm5s2U) in the tRNA wobble led to codon-biased gene-expression alteration in starved animals. Similarly, Magro et al.

49

detected 37

modifications from RNA of E. coli by metabolic labelling and LC-HRMS analysis. It is worth noting

that

a

new

modification 9


of

msms2i6A


Page 10 of 47

(2-methylthiomethylenethio-N6-isopentenyl-adenosine) was confirmed present in E. coli tRNA. Very recently, Huber et al.

50

established stable isotope, dual-labeling approach in

conjugation with LC-MS for the study of RNA modifications. In this report, human HEK293T cells were cultured in the presence of generate

13

13

CD3-L-methionine to metabolically

CD3-m5C in RNA. The medium was then replaced with medium containing

unlabeled methionine and 1,3-15N2-cytidine. The resulting RNA were enzymatically digested to nucleosides and subjected to LC-MS analysis. Using this strategy, they identified a novel derivative of m5C, 2’-O-methyl-5-hydroxymethylcytidine (hm5Cm), in RNA from mammalian cells, tissue and several organisms.

2.3.2 Chemical labeling-mass spectrometry Many modifications generally exist in very low abundance in RNA and traditional LC-MS analysis may not be able to achieve the sensitive detection of these low-abundant modifications. In this respect, chemical labeling strategy has been developed to combine with LC-MS to obtain higher detection sensitivity.51-56 Our group recently developed a novel strategy by oxidation-derivatization combined with LC-MS analysis for determination of hm5C and f5C in both DNA and RNA.57 In this strategy, MnO2 was utilized to convert hm5C to f5C, which was further labeled by dansylhydrazine. Dansylhydrazine harbors the hydrazide moiety that can readily react with aldehyde group in f5C to yield hydrazone derivatives carrying an easily chargeable tertiary ammonium, which enables the increased ionization efficiency of f5C during LC-MS analysis. 10


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Using this method, we reported the presence of f5C in RNA of mammals. In addition, our group established a method using chemical labeling coupled with LC-MS analysis for the simultaneous determination of m5C, hm5C, f5C and ca5C in RNA.58,59 The detection sensitivities of these modifications increased by 70-313 folds upon 2-bromo-1-(4-diethylaminophenyl)-ethanone labeling. We discovered the existence of ca5C in RNA of mammals by the chemical labeling-MS-based analysis. Furthermore, we found the contents of hm5C in RNA of human colorectal carcinoma and hepatocellular carcinoma tissues significantly decreased compared to tumor adjacent normal tissues, suggesting that hm5C in RNA may play functional roles in cancer development and formation. Using the similar analytical strategy, we recently simultaneously detected six formylated nucleosides, including

5-formyl-2’-deoxycytidine,

f5C,

5-formyl-2’-deoxyuridine,

5-formyluridine,

2’-O-methyl-5-formylcytidine and 2’-O-methyl-5-formyluridine, from DNA and RNA of cultured human cells and mammalian tissues.60 Wrobel et al.

61

proposed a strategy enabling sensitive quantification of m5C and other

nucleosides based on LC with inductively coupled plasma mass spectrometry (ICP-MS) detection. The procedure relies on labeling ribose with osmium (Os) by formation of a ternary complex between cis-diol ribose groups. The separation of Os-labeled cytidine (C), uridine (U), m5C and guanosine (G) was achieved on C18 column. The LOD of m5C is 21 pmol/L, indicating a sensitive quantification of modified nucleosides. This study provided quantitative data on RNA cytosine methylation and demonstrated the utility of ICP-MS as complementary analytical tool in study of RNA modifications.

11



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2.3.3 Ion mobility spectrometry-mass spectrometry With the advancement of gas-phase separation technology, Fabris’s group established ion mobility spectrometry-mass spectrometry (IMS-MS) for the direct analysis of RNA modifications.62,63 IMS-MS can separate ions by shape and charge density and add an extra dimension of separation in the gas phase, which enabled the differentiation of the isobaric compounds in the mixture, such as UMP/ΨMP.62 Using IMS-MS, Fabris et al.

63

showed the

comprehensive modification profiles that are unique for different cell types and metabolic states, demonstrating the IMS-MS technique was capable of providing detection and quantification of RNA modifications in complex mixture.

2.3.4 Capillary electrophoresis-mass spectrometry Khan et al.

64

reported a capillary electrophoresis-mass spectrometry (CE-MS) method

for the direct analysis of miRNA from biological samples. miRNA sequences and contents are considered to be associated with certain cancers.65 Using the CE-MS method, the authors detected two endogenous human circulating miRNA, iso-miR-16-5p and miR-21-5p isolated from B-cell chronic lymphocytic leukemia serum. The CE separation with following MS analysis provided label-free quantitation and also revealed modifications of miRNA, including 5’-end phosphorylation, 3’-end adenylation and uridylation. microRNA profiling of serum samples with CE-MS has the potential to be an invasive bioassay for clinical diagnostics.

2.4 Thin layer chromatography 12


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Thin

layer

chromatography

(TLC)

followed

by

UV

spectrophotometric

or

autoradiography detection has been used for more than half century for analysis of RNA components. This method is based on the differences in net charge, polarity and hydrophobicity between nucleotides. The modified nucleotides can usually be determined by assignment to known standards through comparison of their mobility on chromatography.66 TLC is suitable for the analysis of 5’ or 3’-nucleotide monophosphates (NMPs). Compared to one dimension (1D) chromatography, 2D separation on cellulose plates that can greatly improve the separation capability is the most frequent version of TLC. Because the mobility of more than 100 chemically distinct modified nucleotides has been characterized, identification and quantification of 32P-labeled modified nucleotides down to femtomol level was straightforward.16,67 2D-TLC has been widely used to detect various RNA modifications, such as m62A, m6Am, m62Am, ac4Cm, m5Cm, m4C, m4Cm, m2Gm, m22Gm, m2,2,7G and m2,7G.16 Compared to unlabeled samples that generally require microgram of RNA by UV-based detection, radioactive labeling with [32P] can largely increase the sensitivity. As for TLC-based analysis of RNA modifications, there is no need for expensive instrumentation. However, the analytical procedure with radioactive labeling by TLC is relatively time-consuming.

2.5 Fluorescence labeling Torres et al.

68

developed selective derivatization of cytosine moieties with

2-bromoacetophenone for the determination of global DNA cytosine methylation by LC with spectrofluorimetric detection. In fact, this method is also suitable for analysis of RNA 13



modifications such as m5C. The quantification of m5C can be performed without the elimination of DNA since the 2-bromoacetophenone-labeled m5C and m5dC can be well separated in LC. With the 2-bromoacetophenone labeling, the LODs of m5C can reach 16.6 fmol that is comparable to the LC-MS-based detection. Using this fluorescence labeling strategy, the authors successfully evaluated the effects of environmental stresses on DNA and RNA cytosine methylation in living organisms.

3 Location analysis of RNA modifications The aforementioned methods and techniques focus on the overall detection of RNA modifications. However, the location information of modifications in RNA by these techniques cannot be obtained. As deciphering the functions of RNA modifications relies on the determination of the location of RNA modifications, a variety of methods and technologies have been developed to comprehensively map RNA modifications. The mapping approaches for RNA modifications mainly include MS, reverse transcription (RT), chemical labeling/treatment, and immunoprecipitation in combination of subsequent sequencing, or PCR, or gel electrophoresis analysis (Figure 2).

3.1 Mass spectrometry MS has advanced significantly on RNA study over the past decade. The improved analytical methods, protocols and software for analysis of tandem MS (MS/MS) data greatly promote the characterization of modifications in RNA. MS/MS is commonly conducted for sequence analysis of RNA and mapping of RNA modifications.69 Although the methods, 14


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protocols, and databases are insufficient in the RNA field,18 rapid advances have been made in recent years.

3.1.1 Liquid chromatography-mass spectrometry McCloskey’s group initially developed a strategy for location analysis of modifications in RNA by LC-MS.30 RNA was typically digested with RNase T1 or RNase A. The resulting oligonucleotide fragments were then subjected to ion pairing LC-MS/MS analysis, which can generate mass and sequence information, including the sites of modifications on RNA fragments (Figure 3B). Later, a series of studies have been conducted using LC-MS/MS to locate RNA modifications, including 3-methylpseudouridine (m3U) in E. coli 23S rRNA,70 N4-acetylcytidine (ac4C) and N4-acetyl-2’-O-methylcytidine (ac4Cm) in 5S rRNA of Sulfolobus solfataricus and Pyrodictium occultum,71 2-thiouridine (s2U) in mitochondria tRNA of L. tarentolae,72 m5U and Ψ in E. coli tmRNA.73 In addition, a phylogenetic overview was obtained by comprehensive mapping of modifications on 16S rRNA from prokaryotes with LC-MS/MS analysis.74,75 Liu’s group described an approach for the discovery of RNA-small molecule conjugates by combining size-exclusion chromatography and LC-MS analysis.76,77 In this approach, cellular RNA was first subjected to size-exclusion chromatography and the macromolecular fraction (> 2,500 Da) was divided into two halves, which were treated with nuclease P1 or heat-inactivated nuclease P1. Both samples were subjected to size-exclusion chromatography again, and the small-molecule fraction from each was analyzed by high-resolution LC-MS. Using this approach, they successfully identified CoA-linked RNA 76 and NAD-linked RNA 15



77

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at the 5’ terminus in E. coli and S. venezuelae. The isomers of U and Ψ cannot be differentiated from each other by direct LC-MS

analysis because Ψ is a mass-silent modification. By virtue of the different fragmentation behavior of U (N-glycosidic bond) and Ψ (C-glycosidic bond) in RNA upon collisionally activated dissociation, Taucher et al. 78 proposed a LC-MS-based method to locate Ψ in RNA. However, this approach was only demonstrated by standard RNA samples and further improvement is required for the analysis of Ψ in real samples. Recently, Yamauchi et al.

79

developed a novel MS-based method for direct determination of Ψ in RNA. A Ψ-containing RNA fragment is assigned by an accurate measurement of a signature anion ([C9H7N2O4]1−, m/z 207.04), and pseudo-MS3 was used to determine the Ψ-containing nucleotide sequence. By applying this method, they identified all of the known Ψ sites in the canonical spliceosomal snRNA and found two new Ψ sites in the U5 and U6 snRNA of mammalian cells. To further enhance detection sensitivity and improve the coverage of the RNA fragments with modifications, Cao et al.

80

developed a mass-exclusion list strategy for mapping

modifications in RNA. With the removal of unmodified RNA fragments by generating a mass-exclusion list, only oligonucleotides carrying modifications were analyzed to produce MS/MS spectra. The utility of this mass-exclusion list coupled with LC-MS/MS strategy was demonstrated by mapping RNA modifications in total tRNA from prokaryotes. Creation of a mass-exclusion list enhances the number of RNase digestion products by ∼20%, which effectively expanded the MS/MS coverage of digested oligonucleotides carrying modifications and simplified the MS/MS spectra. 16


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Suzuki’s group used chaplet column chromatography system to isolate mitochondrial tRNA from bovine for LC-MS/MS analysis of digested RNA fragments.81 They found 15 species of modifications at 118 positions. Using the similar strategy, recently they further identified 2-methylthio cyclic N6-threonylcarbamoyladenosine (ms2ct6A) as a novel RNA modification at position 37 of tRNA from Bacillus subtilis, plants and Trypanosoma brucei.82

3.1.2 Top-down mass spectrometry In the majority of MS-based RNA studies, long RNA species were enzymatically digested to small size of RNA fragments prior to MS analysis. Analysis of the intact RNA (top-down analysis) is an alternative strategy for the study of modifications on RNA (Figure 3C). One such example is the identification of the presence of 2’-O-methylation on the terminal of microRNA in plant by top-down MS analysis.11 Taucher et al.

83

proposed a direct approach for the characterization of RNA

modifications by top-down MS. They demonstrated 89% sequence coverage in a single collisionally activated dissociation spectrum of E. coli tRNAVal and showed electron detachment dissociation and collisionally activated dissociation data can be combined to provide extensive sequence information for the characterization of RNA modifications. This method can directly provide the types and location of RNA modifications without the need of enzymatic digestion. Very recently, Glasner, et al.

84

utilized top-down MS to characterize

RNA methylations (m6A, m5C, m3U, and m5U) in the mixture of either isomers of RNA or nonisomeric RNA forms. In this method, both collisionally activated dissociation and electron detachment dissociation provided sequence information and can be used for the identification 17



and localization of methylated residues. The top-down strategy currently heavily relies on sophisticated mass spectrometers. In addition, so far only purified samples can be analyzed by top-down strategy. Improvement of methods is essential to address the challenging of the characterization of RNA mixtures for this approach to become widely used in solving biological problems.

3.1.3 Stable isotope labeling-mass spectrometry Recently, Taoka et al.

85

developed a stable isotope labeling method combined with

LC-MS analysis to characterize the sites of RNA modifications. In this method, an unmodified heavy isotope-labeled reference RNA (heavy-G RNA) was synthesized in vitro and added to the sample RNA with an equal amount. The mixture of heavy stable-G RNA and sample RNA was digested and analyzed by LC-MS (Figure 3B). The RNA fragment without modifications co-eluted with the corresponding heavy-G RNA fragment as paired MS signals, but RNA fragment with modifications eluted differently from the corresponding heavy-G RNA fragment and showed characterized MS spectrum for each modification. The site of modification was identified by examining the chromatographic peak that contained only the light fragment. Using this method, 122 modification sites were identified in S. pombe rRNA. This stable isotope labeling-LC-MS-based method has been demonstrated to be a valuable tool for studies of modifications in different RNA species.

3.1.4 MALDI-mass spectrometry Kirpekar’ group proposed an approach to locate modifications on RNA by 18


Page 18 of 47

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matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis.86 RNA was fragmented by specific RNase followed by MALDI-MS analysis (Figure 3B). The spectra were then compared to a theoretical digest of the unmodified RNA. RNA fragments that deviate in m/z from theoretical values are further analyzed by MALDI-MS/MS to locate the modifications. The MALDI-MS-based approach has been used in screening for rRNA modifications.87,88 However, this approach generally is less informative about the identification and location of RNA modifications, and often limits the use to RNA smaller than ~500 nt.36

3.1.5 Chemical labeling-MALDI-mass spectrometry Because Ψ is a mass-silent modification, the normal MALDI-MS analysis cannot locate Ψ in RNA. To resolve this issue, Patteson et al. 89 developed a method using chemical labeling combined with MALDI-MS for the location analysis of Ψ in RNA (Figure 3B). They utilized 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide (CMC) to selectively derivatize Ψ. After CMC labeling, all Ψ residues contain a 252 Da mass tag which can be readily identified. To determine the sequence location of Ψ, MALDI-MS/MS analysis of RNase T1 digestion products before and after CMC labeling were generally used to map the sites of Ψ in RNA. Specific cyanoethylation of N1 in Ψ has been known for many years.90 Mengel-Jùrgensen et al.

91

used MALDI-MS to analyze Ψ in tRNA that was specifically

cyanoethylated by acrylonitrile. The tRNA was first cyanoethylated and then digested with either RNase A or RNase T1. Cyanoethylated digestion fragments were identified by comparing the MS spectra of untreated and acrylonitrile-treated samples, where the addition 19



of one acrylonitrile resulted in a mass increase of 53 Da. The sites of modifications could be identified by MALDI-MS/MS on the cyanoethylated digestion fragment. One Ψ in tRNATyrII from E. coli was then successfully identified by this method. MALDI-MS in combination with cyanoethylation has been demonstrated to be a useful complement to the CMC labeling with MS detection.

3.2 Endonuclease digestion The principle of the restriction endonuclease method is based on the fact that some enzymes can specifically recognize the particular RNA modification. So the resulting patterns can provide a readout of RNA modifications by in combination with various detection methods such as gel electrophoresis, PCR, TLC, and MS. Morse et al.

92

developed a method for investigating the sites of inosines in RNA by

specifically cleaving RNA at inosines using glyoxal treatment followed by RNase T1 digestion. In this strategy, RNA was first treated with glyoxal that can form a stable complex with G. The formed complex was further stabilized by borate. However, inosine cannot react stably with glyoxal. The glyoxal-G-borate complex is resistant to RNase T1 cleavage and only inosine sites were cut by RNase T1. The produced fragments by RNase T1 were then further analyzed by gel electrophoresis or PCR. This method provides a potential way to map inosines in RNA. Yu et al.

93

previously developed a method to locate 2’-O-methylation in RNA using

RNase H digestion. In this strategy, a 2’-O-methyl RNA-DNA chimeric oligonucleotide was used to hybridize to the target RNA followed by RNase H cleavage and gel electrophoresis 20


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analysis. With this method, two sites of 2’-O-methylation, Gm1448 in 18S rRNA and Am394 in 28S rRNA of Xenopus were identified. In addition, the use of site-specific RNase H cleavage has been extended to the detection of other RNA modifications such as Ψ.94 The need of large amount of purified RNA limits the use of endonuclease digestion-based analysis because individual mRNA or lncRNA is too rare to be efficiently purified. In this respect, Liu et al. 95 developed a new method called SCARLET (site-specific cleavage and radioactive-labeling followed by ligation-assisted extraction and thin-layer chromatography) to determine the sites of m6A on mRNA/lncRNA. SCARLET method combines RNase H site-specific digestion, splinted ligation, ribonuclease digestion, and TLC analysis. The m6A status in two human lncRNA and three human mRNA was successfully identified with SCARLET. In addition to m6A, SCARLET can also be used to investigate other RNA modifications, such as m5C, Ψ, and 2’-O-methylation. SCARLET method only requires normal lab instruments, which is readily applicable to researchers for the investigation of RNA modifications.

3.3 Reverse transcription-gel electrophoresis During reverse transcription (RT), some RNA modifications can lead to RT-arrest or misincorporation of a non-complementary dNTP, both of which contribute to the characterized RT signature. The RT-arrest can provide information in the form of truncated cDNA.96 Abortive cDNA or misincorporation in the cDNA sequences can be analyzed by polyacrylamide gel electrophoresis (PAGE), PCR or sequencing. Compared to LC-MS-based method, this strategy doesn’t require the purification of RNA because specific annealing of 21



primers to RNA can achieve selection of target RNA. As both abortive RT and misincorporation signatures are not yet available for the large majority of modifications, their application to the detection of RNA modifications is still limited. However, many RNA modifications were still specifically mapped by the RT-arrest and misincorporation, such as 3-[3-amino-3-carboxypropyl]-1-methylpseudouridine (amΨ) in 18S rRNA of D. melanogaster,97 m1G745 in 23S rRNA of E. coli,98 m62A in 18S rRNA of yeast.99 2’-O-methylation is present in various cellular RNA and is essential to RNA biogenesis. Dong et al.

100

established a method termed RTL-P (reverse transcription at low

deoxyribonucleoside triphosphate concentrations followed by polymerase chain reaction), to detect 2’-O-methylation sites in RNA. The procedure of RTL-P includes a site-specific primer extension by reverse transcriptase at low dNTP concentration followed by semi-quantitative PCR amplification. They then used RTL-P to successfully detect numerous 2’-O-methylation sites in human and yeast rRNA, as well as in mouse piwi-interacting RNA (piRNA). The RTL-P method has been demonstrated to be a useful platform for the systematic analysis of 2’-O-methylation in diverse RNA species.

3.4 Reverse transcription-sequencing The RT-arrest induced truncated cDNA or misincorporation in cDNA sequences can also be analyzed by subsequent next-generation sequencing, which provides more comprehensive analysis for mapping RNA modifications. m1A, m1G, and m3C, are prevalent modifications in tRNA and play important roles in the 22


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biogenesis, stability and functionality of tRNA. Recently, Cozen et al.

101

described

AlkB-facilitated RNA methylation sequencing (ARM-seq) using the property that these modifications can cause RT-arrest to map their sites in tRNA. In ARM-seq, removal of m1A, m1G, and m3C by AlkB treatment provided the full-length cDNA; however, the untreated RNA produced truncated cDNA. Therefore, these methylation sites in RNA can be obtained by comparison of reads in treated versus untreated samples. The comparative methylation analysis using ARM-seq provided the first comprehensive mapping of m1A, m1G, and m3C in small RNA derived from tRNA. More recently, Hong et al.

102

developed an antibody-independent method to localize

m6A at single-nucleotide resolution via 4SedTTP incorporation and FTO demethylation. 4SedTTP can base pair with adenine stably but impair the formation of m6A-T pairing. Therefore, the truncation in m6A site is formed during reverse transcription using canonical A, C, G and 4SedTTP. The RT stop signals of RNA with or without FTO treatment were then compared to determine the sites of m6A by subsequent sequencing. This approach is promising in transcriptome-wide mapping of m6A in single-nucleotide resolution in the future. Despite these advantages, the reverse transcription for detection of modifications in RNA still has some limitations. The RT-based detection method can be affected by the inherent properties of modifications and the RNA structures. Therefore, the presence of an arrest or misincorporation in the primer extension profile does not necessarily imply the presence of a modification. In addition, the degradation of RNA may also complicate the analysis of the data. Thus, multiple methods are necessary to confirm the results obtained by RT-based analysis. 23



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3.5 Reverse transcription-single molecule real-time detection Vilfan et al.

103

developed a method for locating m6A in RNA by single-molecule

real-time (SMRT) detection upon reverse transcription. By monitoring the kinetics of reverse transcriptase that synthesizes cDNA in real time, information about sequence content and sites of modifications in RNA can be obtained. The kinetics parameter of reverse transcriptase on the m6A-containing RNA is distinctly different with that on the control RNA in the SMRT analysis, with the frequency of pulse at the m6A position being obviously decreased compared to the canonical A. This characterized frequency of pulses for each type of nucleoside was then used to distinguish m6A with other nucleosides. In theory, this method is also suitable for the analysis of sites of many other modifications in addition to m6A in RNA. However, analysis of modifications from RNA by SMRT currently has only been realized by synthetic RNA carrying modifications, and much effort is still required to achieve the mapping of modifications in real RNA samples by SMRT.

3.6 Chemical labeling-reverse transcription RT-based mapping of RNA modifications can be considerably improved by means of chemical labeling. In this strategy, chemical reagents that can specifically react with a given modification are carefully selected to enhance the characterized RT-arrest or misincorporation event. The RT profile obtained after chemical modifications generally allows identification of the modifications and their location in RNA sequence. In this respect, Bakin et al.

104

used

CMC (N-cyclohexyl-N’-β-(4-methylmorpholinium)ethylcarbodiimide p-tosylate) to modify 24


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U-like and G-like residues. Under alkaline conditions, only CMC-Ψ complex is stable, thus leading to the reverse transcription stop at Ψ. With this method, four new Ψ sites in 23S RNA of E. coli were discovered by subsequent PAGE analysis. Herzog et al.

105

developed an orthogonal chemistry approach based on iodoacetamide

(IAA) labeling to detect s4U at single-nucleotide resolution by reverse transcription-dependent thymine-to-cytosine conversions. The thiol-reactive compound IAA was labeled to s4U and the formed alkylated s4U preferably pairs with G than A, which induced the thymine-to-cytosine conversions. This mutation signature of IAA labeled s4U can be used for the analyze s4U at single-nucleotide resolution.

3.7 Chemical labeling-sequencing Adenosine-to-inosine (A-to-I) editing that occurs in a variety of biological processes is prevalent in RNA.106 However, inosine cannot be directly detected by reverse transcription due to its base-pair with cytidine. Specifical chemical labeling with RNA modifications may change their RT signature by RT arrest or misincorporation. In this respect, inosine-specific cyanoethylation by acrylonitrile generates RT stop, allowing A-to-I conversion sites to be obtained by subsequent sequencing analysis. Along this line, Sakurai et al.

107

developed

inosine chemical erasing (ICE) method to map inosines in mRNA based on cyanoethylation combined with reverse transcription-sequencing. In this method, acrylonitrile was used to cyanoethylate inosine to form N1-cyanoethylinosine (ce1I), which can destroy the base-pairing with cytidine and then lead to an arrest in reverse transcription. Untreated inosines were identified as guanosines in sequencing. By combining ICE with sequencing, the 25



Page 26 of 47

authors identified 5,072 inosine sites in mRNA of human brain tissue. More recently, Yi’s group developed N3-CMC-enriched Ψ sequencing (CeU-seq) based on selective chemical labeling and pulldown of Ψ to map Ψ sites in human transcripts (Figure 4).108 In this method, N3-CMC was synthesized to specifically react with the Ψ on RNA, then biotin was subsequently conjugated to the N3-CMC-Ψ RNA via click chemistry. The biotin-CMC-Ψ RNA was enriched using streptavidin beads followed by next-generation sequencing analysis. Because the biotin-CMC-Ψ complex can cause the arrest one base downstream

of

Ψ

in

reverse

transcription,

this

CeU-seq

method

can

offer

single-base-resolution mapping of Ψ. With this method, 2,084 Ψ sites in 1,929 human transcripts were identified. Collectively, this strategy enables comprehensive analysis of transcriptome-wide pseudouridylation and provides a valuable tool for study of Ψ-mediated epigenetic regulation.

3.8 Chemical conversion-sequencing Bisulfite sequencing has been widely used in mapping m5C in DNA. Similarly, a study using bisulfite conversion with a modified procedure followed by primer extension was successfully established to map m5C sites in yeast tRNAHis.109 Combination of RNA bisulfite conversion with sequencing was also reported for the location analysis of m5C in both rRNA and tRNA of Drosophila.110 Later, Squires et al.

111

employed the bisulfite conversion coupled with next-generation

sequencing to map m5C in human mRNA and non-coding RNA. They discovered 10,275 m5C sites in mRNA and non-coding RNA. Particularly, m5C was found to be enriched in the 26


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untranslated regions and near Argonaute binding regions in mRNA. In addition, Edelheit et al. 112

also detected m5C sites in archaeal mRNA and identified a consensus motif of

AUCGANGU that directs methylation in S. solfataricus using bisulfite conversion coupled with next-generation sequencing. These studies demonstrated the widespread existence of m5C in coding and non-coding RNA, suggesting a regulatory role of m5C in RNA functions. RNA bisulfite sequencing typically suffers from incomplete deamination of canonical cytidines, which may produce false positives. In addition, because RNA is prone to degradation under alkaline condition, the bisulfite conversion need to be carefully optimized. 2’-O-methylation, the most abundant modification in rRNA, plays critical roles in ribosome assembly and translation fidelity.113 Birkedal et al.

114

developed a alkaline

hydrolysis combined with next-generation sequencing method (RiboMeth-seq) to map 2’-O-methylation sites in yeast rRNA. RiboMeth-seq is based on the activation of the 2’-OH of the ribose sugar for nucleophilic attack on the neighboring phosphodiester bond under alkaline condition, which results in strand cleavage. On the contrary, the 2’-O-methylated ribose is resistant to alkaline cleavage. Using this method, the authors realized the comprehensive mapping of 2’-O-methylation in rRNA. Later on, Krogh et al.

115

applied the

same method to map 2’-O-methylation and found 106 sites in rRNA from HeLa cells. However, RiboMeth-seq method is less effective to detect 2’-O-methylation sites in mRNA. To address this challenge, Dai et al.

116

recently developed Nm-seq method for

transcriptome-wide mapping of 2’-O-methylation with single-base-resolution by virtue of the differential reactivity of 2’-O-methylated and 2’-OH nucleotides to periodate oxidation. Typically, RNA was fragmented and subjected to oxidation-elimination-dephosphorylation 27



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cycles, which can remove 2’-OH nucleotides from 3’ to 5’ to expose internal 2’-O-methylated sites at 3’ ends of fragments. The resulting RNA were then analyzed by next-generation sequencing. Using this method, they discovered thousands of 2’-O-methylation sites in mammalian mRNA with 2’-O-methylation sites being mainly enriched in CDS region.

3.9 Immunoprecipitation-sequencing RNA modifications generally exist in low-abundance. Thus, enrichment of RNA fragments that carry modifications before sequencing analysis is essential for effective mapping of modifications. The recent development of RNA immunoprecipitation followed by next-generation sequencing (RIP-seq) offers a promising platform for transcriptome-wide analysis of RNA modifications. RIP-seq heavily depends on specific antibodies to enrich the targeted modifications. Meyer et al. m6A-specific

117

RNA

established a method for transcriptome-wide mapping of m6A by immunoprecipitation

coupled

with

next-generation

sequencing

(MeRIP-seq). Using this method, they identified mRNA of 7,676 genes that contain m6A in mammal. m6A sites were found to be mainly enriched near stop codons and in 3’ UTRs. In addition, Dominissini et al.

118

used MeRIP-seq to achieve a transcriptome-wide mapping of

human and mouse m6A modification. Over 12,000 m6A sites were identified in mRNA of more than 7,000 human genes. In addition to mammals, 1,308 m6A sites in 1,183 mRNA were also identified in yeast by using MeRIP-seq.119 These studies provided a resource for locating m6A in transcripts and revealed the regulatory roles of m6A in mRNA. However, RIP-seq approach only can localize m6A sites in RNA in a region of 100-200 28


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nt long and cannot achieve single-base-resolution mapping of m6A due to the inherent shortcoming of immunoprecipitation.13 Covalent crosslinking of antibodies to modifications of RNA can generate specific RT signature, which may allow single-base-resolution analysis of m6A by subsequent next-generation sequencing. For example, Linder et al.

120

developed

an approach of m6A single-base-resolution cross-linking and immunoprecipitation (miCLIP). In this method,

specific mutation at m6A sites can be obtained after UV light-induced

antibody-RNA cross-linking and reverse transcription. They found that these antibodies also induced mutational signature at N6,2’-O-dimethyladenosine (m6Am), a modification found at the first nucleotide of certain mRNA. Using this method, m6A and m6Am were successfully mapped at single-base-resolution in human and mouse mRNA and small nucleolar RNA (snoRNA). In addition to m6A, N1-methyladenosine (m1A) is another prevalent RNA modification.121 Recently, Yi’s group developed the m1A-ID-seq technique for transcriptome-wide mapping of m1A based on m1A immunoprecipitation and the inherent ability of m1A to stall reverse transcription.122 With m1A-ID-seq method, 901 m1A sites were identified in mRNA and noncoding RNA with m1A being mainly enriched in 5’ untranslated region of mRNA. In the meantime, using the similar strategy, Dominissini et al.

123

found that m1A was enriched

around the start codon and was dynamic in response to physiological conditions. In this study, a fraction of RNA fragments was treated to generate m1A-to-m6A rearrangement before cDNA synthesis. By comparing the readouts of both m1A and m6A, single-base-resolution detection of m1A was accomplished. Collectively, this approach allows comprehensive location analysis of m1A and provides valuable platform for functional studies of RNA 29



modifications.

3.10 Nanopore Besides the next generation sequencing-based methods, the innovative third-generation sequencing techniques also show promising in mapping of RNA modifications. In recent years, nanopore-based technology has emerged as a powerful approach in nucleic acids sequencing.124 A voltage is applied to monitor the changes of the ionic current while molecules pass through the nanometre-scale pore,125 which can differentiate modified nucleosides from canonical nucleosides. Due to the inherent advantage, nanopore-based sequencing has the potential to globally characterize RNA modifications. Ayub et al.

21

established a method for the RNA base recognition in immobilized

oligonucleotides using α-hemolysin (αHL) nanopore. Characterized current distributions were achieved to enable distinct discrimination of the four nucleobases, guanine, cytosine, adenine, and uracil, in RNA. In addition, the modified nucleobases, such as inosine, m6A, and m5C, also can be distinguished. The nanopore-based analysis offers a promising strategy for the unlabeled and real-time analysis of the sites of modifications in RNA. Though the method has not yet been applied to the global profiling of real RNA samples, one can envision an array of chip-based nanopore that allows parallel sequencing and detection of RNA modifications.

3.11 Cryo-electron microscopy Cryo-electron microscopy is increasingly becoming an important technology for deciphering three-dimensional structures of biological specimens in a near-native state.126 30


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Recently, Natchiar et al. 127 successfully visualized more than 130 individual modifications in human rRNA using cryo-electron microscopy (Figure 5). A resolution of approximately 2.5 Å was achieved in most of the region of ribosome, which allowed the unambiguous determination of the chemical structures. The atomic model of the human ribosome by cryo-electron microscopy provided many fine details of modifications in rRNA. Therefore, many new rRNA modification sites were identified in this study. The high-resolution structure of human ribosome may also promote understanding the roles of rRNA modifications in human diseases.

3.12 Ligation-assisted analysis The RNA modifications may affect the molecular recognition and then the subsequent enzymatic reactions. Based on this principle, Dai et al

128

established ligation-based sites

analysis of Ψ and m6A in RNA. In this strategy, the modifications of Ψ and m6A in RNA can affect the ligation of two complementary oligo DNAs by T4 DNA ligase, which was used to discrimination of modifications and canonical nucleobases in RNA. Similarly, Liu et al.

129

recently developed a DNA ligase-based method for sensitive detection of m6A at single-nucleotide resolution. Two DNA probes were ligated using the targeted RNA template with T3 DNA ligase. The presence of m6A in RNA can largely compromise the ligation efficiency of the two DNA probes. The ligated products were then amplified with real-time qPCR. By comparing the real-time fluorescence curves, m6A in RNA can be determined. The ligation assisted approach can be readily used to quantify the extent of modification at defined positions. 31



4 Conclusions and perspectives Innovations in analytical technology have greatly promoted the study of RNA modifications in the past several years. Apart from better detection sensitivity and accuracy, comprehensive detection and mapping of different modifications in the same biological sample are desirable. Challenges arising from the needs of tools for deciphering the functions of RNA modifications will continue to stimulate the development of analytical methods and software. It is important to keep in mind the advantages and disadvantages of any analytical techniques. We expect MS will continue to play an important role in the investigation of RNA modifications. MS is powerful in its ability to discover new low-abundant modifications, to detect a broad range of modifications in a single analysis, and to locate modifications on RNA. While MS cannot compete with the throughput of next-generation sequencing technology. However, should proteomics-like strategy and platform for mapping of RNA modifications be developed, MS-based mapping of modifications in different RNA species is greatly feasible. Further investigation of the differential chemical reactivity of modifications with a variety of chemical reagents to be coupled with high-throughput sequencing is still a promising direction for mapping RNA modifications. A variety of chemical reactions appropriate for modified nucleosides are available if we revisit the chemistry of nucleic acids. In this situation, one can envision a direct detection of multiple RNA modifications by modification-specific derivatization in combination with high-throughput sequencing. High-throughput RNA-seq methods have proven worthwhile in global profiling of given 32


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RNA modifications. This technology is still, however, limited to only several of the prevalent modifications in RNA. The inherent error in the analysis can lead to false positive or negative results, which impacts the accuracy of assigning modification sites especially for those of low-abundant modifications. Increasing the read length and sequence depth would be significant improvements. High-throughput RNA-seq right now cannot match the ability of MS to identify many modifications present in a single sample. Rapidly-evolving third-generation sequencing technologies, such as SMRT sequencing and nanopore sequencing, hold great promise on RNA modifications study. The characterized RT signatures of modifications in SMRT sequencing can be analyzed by single molecule spectroscopy. Nanopore sequencing technology has been explored in DNA modifications field, and a similar development in RNA sequencing seems promising, although currently is still in the early stage. The application of third-generation sequencing technologies for the mapping of modifications in real RNA samples may take some time in coming. The number of chemically distinct modifications is still growing and the known modifications may also occur in new sites of RNA species. Whichever method is used, identification of modifications and mapping their sites in RNA molecules remain difficult. Particularly, mapping study of RNA modifications in the transcriptome is still at its infancy, and we expect that input from the analytical chemistry field will continuously promote and enrich our understanding of RNA modifications.

33



Biography Bei Chen is a Ph.D. student in the Department of Chemistry, Wuhan University, China. She received her BSc degree in Chemistry at Wuhan University in China. Presently, she is working under the guidance of Prof. Bi-Feng Yuan, focusing on the development and applications of analytical methods for the study of nucleic acid modifications.

Biography Bi-Feng Yuan studied biochemistry and biophysics at Wuhan University in China, where he received his BSc and Ph.D. degrees in 2001 and 2006, respectively. From 2006-2007 and 2007-2011, he worked as a postdoctoral researcher in the Department of Biological Sciences at National University of Singapore and in the Department of Chemistry at University of California Riverside, respectively. He joined Wuhan University in 2011 and is currently a Professor of Chemistry. His research focuses on the development and application of new analytical techniques in the investigation of the occurrence, location, and biological functions of nucleic acid modifications.

Biography Yu-Qi Feng received his BSc and Master’s degrees from Lanzhou University in China in 1982 and 1985, respectively. From 1986 to 1991, he worked in Central China Normal University in China. He then studied chemistry at Chiba University in Japan, where he received his Ph.D. degree in 1996. He joined Wuhan University in 1996 and became a full professor in 2000. His research focuses on the sample pretreatment and LC/MS-based 34


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metabolomics.

AUTHOR INFORMATION Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

Acknowledgements The authors thank the financial support from the National Natural Science Foundation of China (21522507, 21672166, 21721005, 21728802).

35



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Figure legends Figure 1. Schematic illustration of the methods for the overall analysis of RNA modifications.

Figure 2. Schematic illustration of the strategies for the location analysis of RNA modifications.

Figure 3. Schematic illustration of mass spectrometry-based analysis of RNA modifications. (A) Overall analysis of RNA modifications by mass spectrometry. (B) Location analysis of RNA modifications by mass spectrometry. (C) Location analysis of RNA modifications by top-down mass spectrometry.

Figure 4. Schematic illustration for mapping Ψ in mRNA by CeU-Seq. Ψ-containing RNA is enriched by a pulldown step. The biotin-N3-CMC-Ψ cause RT-arrest. The resulting cDNA is then circularized, linearized, PCR amplified and subjected to high-throughput sequencing. Reprinted by permission from Macmillan Publishers Ltd: NAT CHEM BIOL, Li, X.; Zhu, P.; Ma, S.; Song, J.; Bai, J.; Sun, F.; Yi, C. Nat Chem Biol 2015, 11, 592-597 (ref#108). Copyright 2015

Figure 5. Chemical modifications identified in the 60S ribosomal subunit by Cryo-electron microscopy. Reprinted by permission from Macmillan Publishers Ltd: NATURE, Natchiar, S. K.; Myasnikov, A. G.; Kratzat, H.; Hazemann, I.; Klaholz, B. P. Nature 2017, 551, 472-477 (ref#127). Copyright 2017. 41



Figure 1.

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Figure 2.

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Figure 3.

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Figure 4.

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Figure 5.

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