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Chemical tagging for sensitive determination of uridine modifications in RNA†

The discovery of dynamic and reversible modifications in messenger RNA (mRNA) is opening new directions in RNA modification-mediated regulation of biological processes. Methylation is the most prevalent modification occurring in mRNA and the methyl group is mainly decorated in the adenine, cytosine, and guanine base or in the 2′-hydroxyl group of ribose. However, methylation of the uracil base (5-methyluridine, m5U) has not been discovered in mRNA of eukaryotes. In the current study, we established a method of N-cyclohexyl-N′-β-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) labelling coupled with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for the sensitive determination of uridine modifications in RNA. Our results demonstrated that the detection sensitivities of uridine modifications in RNA increased up to 1408 fold upon CMCT labelling. Using the developed method, we identified the distinct existence of m5U in mRNA of various mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methyl group of m5U originated from S-adenosyl-l-methionine (SAM). Our study expanded the list of modifications occurring in mRNA of mammals. Future work on transcriptome-wide mapping of m5U will further uncover the functional roles of m5U in mRNA of mammals.


Introduction
Chemical modication in nucleic acids can delicately regulate gene expression. 1 The functional roles of DNA modications in regulation of gene expression have been well established. 2 However, how RNA modications affect translation and eventually tune gene expression has just begun to be understood. 3 The discovery of a variety of modications in RNA broadens our views on the structures and functions of RNA. 4 To date, over 150 chemically distinct modications have been identied in various species of RNA in all three kingdoms of life. 5,6 Most of these modications are characterized in the abundant ribosomal RNA (rRNA) and transfer RNA (tRNA), where they can alter the RNA secondary structure by affecting base stacking or changing hydrogen bonding patterns. 7,8 RNA modications are found to be involved in various RNA processing and metabolism procedures, including alternative splicing, stability, translation, folding and molecular recognition. [7][8][9][10] The discovery of dynamic and reversible modi-cations in messenger RNA (mRNA) is opening new directions in RNA modication-mediated regulation of biological processes. 11 The technical breakthroughs led to the discovery of a series of modications in eukaryotic mRNA, including N 6methyladenosine (m 6 A), N1-methyladenosine (m 1 A), N 6 ,2 0 -Odimethyladenosine (m 6 Am), 5-methylcytidine (m 5 C), N3-methylcytidine (m 3 C), N7-methylguanosine (m 7 G), 2 0 -O-methylation, inosine (I), and pseudouridine (J). [11][12][13] Very recently, N 4 -acetylcytidine was newly discovered to be present in mRNA of mammals. 14 These studies demonstrated that the modications play versatile roles in mRNA processing and eventually affect the epitranscriptomic state of cells. 15 Methylation is the most prevalent modication occurring in mRNA, such as m 6 A, m 1 A, m 6 Am, m 5 C, m 3 C, and m 7 G, and 2 0 -Omethylation. 11 The methyl group is mainly decorated in adenine, cytosine, and guanine base or in ribose. However, methylation of the uracil base (5-methyluridine, m 5 U) has not been discovered in mRNA of eukaryotes. m 5 U is a common modication found in rRNA and tRNA. 16 Two m 5 U sites were previously identied at positions 747 and 1939 in E. coli 23S rRNA, and one m 5 U site was found at position 54 in E. coli tRNA. 17,18 One early study showed that m 5 U was also present in the tRNA-like domain of tmRNA in E.coli cells. 19 It was proposed that TrmA protein can methylate U54 in the T-arm of most E. coli tRNAs. 18,20 And the RumA protein was responsible for the methylation of U1939 in E. coli 23S rRNA to form m 5 U that may play an important role in translation. 17 But the existence of m 5 U in mRNA of eukaryotes remains elusive.
In eukaryotic cells, the RNA pool predominantly consists of rRNA ($80%) and tRNA ($15%), whereas mRNA only constitutes a small fraction (<5%). 21 The modications in mRNA generally exist in low abundance. 13 For example, m 3 C is present at a frequency around $1 to 5 modications per 10 5 cytidine. 22 Therefore, the low in vivo contents can lead to the detection of modications in mRNA being a challenging task. The mass spectrometry (MS)-based analytical platform has been widely employed for determining modications due to its superior detection sensitivity and powerful capability in the identication of the chemical structures of modications. [23][24][25][26][27][28] However, the ionization efficiencies of uridine and uridine modications in electrospray ionization (ESI)-MS are much poorer than those of other canonical nucleosides and their corresponding modi-cations, 29,30 which makes the detection of uridine modications more difficult.
The chemical labelling strategy that can introduce an easily ionizable functional group into analytes has been developed to improve the detection sensitivities of compounds while in combination with mass spectrometry analysis. [31][32][33] In this respect, the reagent N-cyclohexyl-N 0 -b-(4-methylmorpholinium) ethylcarbodiimide p-toluenesulfonate (CMCT) was previously used to label J and U followed by treatment under alkaline conditions to differentiate J from U. [34][35][36] The CMCT group can stably link to uridine and uridine modications if omitting the alkaline treatment, which then can be utilized to detect the lowabundant uridine modications since the introduction of the permanently positively charged quaternary ammonium group from CMCT can enhance the ionization efficiency of uridine modications. In the current study, we established a method of CMCT labelling coupled with liquid chromatographyelectrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis for sensitive determination of the uridine modications in RNA. Our results demonstrated that the detection sensitivities of uridine modications in RNA increased by 6-1408 fold upon CMCT labelling. Using the developed method, we identied, for the rst time, the existence of m 5 U in mRNA of mammalian cells.

Biological samples and stable isotope labelling tracing
Human embryonic kidney 293T cell line (HEK293T), human cervical carcinoma cell line (HeLa), human hepatic cell line (HL-7702), and human liver carcinoma cell lines (HepG2) were obtained from the China Center for Type Culture Collection. HEK293T, HeLa, and HepG2 cells were grown in DMEM medium supplemented with 10% fetal bovine serum, 100 U mL À1 penicillin, and 100 mg mL À1 streptomycin at 37 C in a 5% CO 2 atmosphere. HL-7702 cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL À1 penicillin, and 100 mg mL À1 streptomycin at 37 C in a 5% CO 2 atmosphere. Male wild-type C57BL/6J mice (2 months old) were obtained from the Center for Animal Experiment/ABSL-3 Laboratory of Wuhan University and sacriced to collect tissues. This study was performed in strict accordance with the NIH guidelines for the care and use of laboratory animals (NIH Publication No. 85-23 Rev. 1985) and was approved by the Animal Care and Use Committee of Wuhan University (Wuhan, China).
As for stable isotope labelling tracing monitored by mass spectrometry, HEK293T cells were cultured in DMEM medium supplemented with 0.3 mM of D 3 -Met to metabolically label the RNA with the CD 3 group. The cells were collected aer culturing for 72 h and then mRNA was extracted.

Purication and enzymatic digestion of different species of RNA
Total RNA was extracted using an E.Z.N.A. HP Total RNA kit (Omega Bio-Tek Inc., Norcross, GA, USA) according to the manufacturer's recommended procedure. Small RNA (<200 nt) was puried using the E.Z.N.A. miRNA kit (Omega Bio-Tek Inc., Norcross, GA, USA). For mRNA isolation, the obtained large RNA (>200 nt) aer small RNA depletion using the E.Z.N.A. miRNA kit was further extracted twice using a PolyATtract mRNA isolation system (Promega, Madison, WI, USA). The resulting mRNA was further processed with agarose gel electrophoresis-based purication, which can remove the potential trace level of contamination of small RNA (<200 nt). The detailed procedure of agarose gel electrophoresis-based purication can be found in the ESI. † The rRNA was puried from the obtained large RNA (>200 nt) by means of sizeexclusion chromatography (SEC) on a Bio SEC-5 column (5 mm, 1000Å, 4.6 Â 150 mm; Agilent Technologies, Foster City, CA, USA) according to a previous study. 37 Isocratic separation was performed at a ow rate of 0.35 mL min À1 at 35 C with 100 mM NH 4 Ac (pH 7.0) as the mobile phase. The fraction was collected and further processed with the E.Z.N.A. miRNA kit to remove NH 4 Ac. The purication was repeated twice to obtain high purity of rRNA.
Enzymatic digestion of RNA was performed according to a previously described method under neutral conditions. 37 Briey, 3 mL of 10 Â buffer (500 mM Tris-HCl, 100 mM NaCl, 10 mM MgCl 2 , 10 mM ZnSO 4 , pH 7.0), 1 mL of S1 nuclease (180 U mL À1 ), 1 mL of venom phosphodiesterase I (0.001 U mL À1 ), and 1 mL of calf intestinal alkaline phosphatase (30 U mL À1 ) were added to the RNA (0.2-5 mg) dissolved in 24 mL H 2 O. The mixture was incubated at 37 C for 4 h. 270 mL of H 2 O and 300 mL of CHCl 3 were added to the resulting solution followed by vortexing for 3 min and centrifugation at 12 000 rpm for 10 min. The upper aqueous phase was collected. The extraction process was repeated 3 times to fully remove the proteins. The aqueous phase was collected and lyophilized and then subjected to subsequent analysis.

Synthesis of
was synthesized from 1-cyclohexyl-3-(2-(4morpholinyl)ethyl) carbodiimide (CME) and D 3 -iodomethane (CD 3 I) according to a previous report 38 with slight modication. The synthesis route is shown in Fig. S1 in the ESI. † Briey, 40 mL of CME (50 mM in ACN) was incubated with 10 mL of CD 3 I at 30 C for 15 h. Then 100 mL of borate buffer (50 mM, pH 8.5) and 200 mL of diethyl ether were added to the mixture. The mixture was separated into a lower aqueous phase and an upper diethyl ether. The unreacted CME and CD 3 I remained in the diethyl ether phase while the newly synthesized D 3 -CMCT was in the aqueous phase. Aer removing the diethyl ether phase, the aqueous phase was collected and used for the labelling reaction.

Chemical labelling
CMCT and D 3 -CMCT were used to label uridine and uridine modications. The labelling reaction was carried out in borate buffer (50 mM, pH 8.5). The reaction conditions, including reaction time and temperature and the concentration of CMCT, were optimized to achieve the best labelling efficiency. Aer the labelling reaction, the mixture was subjected to CeO 2 solid phase extraction (SPE) to remove excess CMCT according to a previously described procedure, 39 and the resulting derivatives were then subjected to LC-ESI-MS/MS analysis. The detailed procedure for the extraction of CMCT labelled nucleosides by CeO 2 SPE is shown in the ESI. †

Analysis of CMCT\D 3 -CMCT labelled uridine modications by LC-ESI-MS/MS
Analysis of CMCT\D 3 -CMCT labelled uridine modications was performed on an AB 3200 QTRAP mass spectrometer (Applied Biosystems, Foster City, CA, USA) equipped with an electrospray ionization (ESI) source (Turbo Ionspray) and a Shimadzu LC-20AD HPLC system (Tokyo, Japan). Data acquisition and processing were performed with AB SCIEX Analyst 1.5 soware (Applied Biosystems). ESI-MS was conducted in positive-ion mode and multiple-reaction monitoring (MRM) was employed for the detection. To achieve the best performance, the mass spectrometric parameters were optimized, and the optimized MS conditions are listed in Table S1 in the ESI. † The separation of CMCT labelled uridine modications was performed on an Inersil ODS-3 reversed-phase column (5 mm, 2.1 Â 250 mm; GL Sciences Lnc., Tokyo, Japan). NH 4 HCO 3 (2 mM, pH 4.5, solvent A) and ACN (solvent B) were used as the mobile phases. Gradients of 0-3 min, 5% B; 3-20 min, 5-40% B; 20-29 min, 40% B; 29-31 min, 40-5% B; and 31-45 min, 5% B were used. The ow rate was set at 200 mL min À1 . Quantication of uridine modications is shown in the ESI. †

High-resolution mass spectrometry analysis
The CMCT\D 3 -CMCT labelled uridine modications were also examined with a high-resolution LTQ-Orbitrap Elite mass spectrometer (Thermo-Fisher Scientic, Waltham, MA, USA) equipped with an electrospray ionization (ESI) source and a Dionex Ultimate 3000 UPLC system (Thermo-Fisher Scientic, Waltham, MA, USA). The LC separation conditions were the same as those for the AB 3200 QTRAP mass spectrometer system. Full MS scans were acquired in positive-ion mode at a resolution of 60 000. Collision induced dissociation (CID) with a collision energy of 25-35 eV was used. The fragments were acquired with a mass range of m/z 200-700 at a resolution of 60 000. The source and ion transfer parameters applied were as follows: heater temperature, 300 C; capillary temperature,

Analysis of mRNA by high-throughput sequencing
The isolated mRNA was processed using a KAPA Stranded RNA-Seq Library Prep Kit (Illumina) for sequencing library construction following the manufacturer's protocol. RNA-seq was carried out on an Illumina HiSeq 4000 (Illumina) platform by Aksomics Inc (Shanghai, China). Detailed information can be found in the ESI. †

Results and discussion
Chemical labelling CMCT carries a carbodiimide group that can selectively react with the NH group at the N3 position of the uracil base (Fig. 1A). The introduction of a constant positively charged quaternary ammonium group from CMCT can efficiently enhance the ionization efficiency of the derivatives in electrospray ionization (ESI)-MS. To achieve good accuracy for quantication and identication, we also synthesized D 3 -CMCT (Fig. S1 in the ESI †). The highresolution mass spectrometry analysis demonstrated that D 3 -CMCT was successfully synthesized (Fig. S2 in the ESI †).
We then used 8 standards of uridine and uridine modications to evaluate the CMCT/D 3 -CMCT labelling reaction (Fig. 1B). Shown in Fig. 2 are the extracted-ion chromatograms and the fragment ions of the CMCT/D 3 -CMCT labelled uridine and m 5 U. The measured precursor ions and product ions of CMCT/D 3 -CMCT labelled uridine and m 5 U were identical to the theoretical values, suggesting that uridine and m 5 U were successfully labelled and the desired derivatives were obtained. The fragmentation analysis demonstrated that the other 6 uridine modications (J, m 1 J, m 6 U, hm 5 U, mo 5 U, and mcm 5 U) were also successfully labelled with CMCT/D 3 -CMCT (Fig. S3 in the ESI †). In addition, the CMCT/D 3 -CMCT labelled uridine modications were further conrmed by highresolution mass spectrometry analysis (Fig. S4 in the ESI †).
It is worth noting that protonated molecules generally undergo fragmentation based on a charge-directed mechanism. But when the charge is xed at one specic atom, charge-remote fragmentation may occur, 41,42 which is caused by hydrogen transfer instead of proton transfer among different atoms of the precursor ion. 43,44 For example, CMCT labelled m 5 U is constantly and positively charged due to carrying a quaternary ammonium group and CMCT labelled m 5 U undergoes charge-remote fragmentation to generate the fragment ion of m/z 252.2 ( Fig. 2C and S5 in the ESI †). CMCT labelled m 5 U can also lose a neutral fragment of 4-methylmorpholine and generate the fragment ion of m/z 409.2, which can further lose a ribose to form the fragment ion of m/z 277.2 ( Fig. 2C and S5 in the ESI †).
It can be seen that the fragment ion of m/z 252.2 is the predominant fragment ion of CMCT labelled uridine modications ( Fig. 2 and S3 in the ESI †). We examined the fragmentation pathway of the CMCT labelled m 5 U with different collision energies ranging from 1 eV to 30 eV. The result showed that the ion of m/z 252.2 was the predominant fragment ion under different collision energies (Fig. S6 in the ESI †). However, we did not observe the cleavage of the N-glycosidic bond that would produce the fragment ion of m/z 378.2 (Fig. S6 in the ESI †). These results suggested that the N-C bond between nucleosides and CMCT was more prone to break than the Nglycosidic bond.
To obtain good labelling efficiency, we optimized the reaction conditions, including reaction temperature and time and the concentrations of CMCT. The result showed that the best labelling efficiencies were obtained at 40 C for most of the uridine modications (Fig. S7A in the ESI †). Therefore, 40 C was used as the reaction temperature. We next optimized the reaction time. The results demonstrated that 14 h of reaction was sufficient for the CMCT labelling ( Fig. S7B in the ESI †). As for the optimization of the concentration of CMCT, the results showed that the signals of CMCT labelled uridine modications reached a plateau when the concentration of CMCT was 50 mM (Fig. S7C in the ESI †). Taken together, the optimized CMCT labelling conditions for uridine modications were 40 C for 14 h with 50 mM CMCT. Under the optimized reaction conditions, more than 91% of uridine modications could react with CMCT to form their corresponding derivatives (Table S2 in the ESI †). And the formed derivatives also exhibited good stability, which was suitable for the subsequent analysis by LC-ESI-MS/ MS (Fig. S7D in the ESI †).

CMCT labelling improves the detection sensitivities of uridine modications
We next evaluated the detection sensitivities of the uridine modications upon CMCT labelling. The CMCT labelled uridine modications were extracted with CeO 2 to remove the excess CMCT followed by LC-ESI-MS/MS analysis (Fig. 3). The results showed that the retention of uridine modications without CMCT labelling on the reversed-phase column was relatively weak (Fig. 4A). However, the retention greatly increased aer CMCT labelling ( Fig. 4B), which is due to the enhanced hydrophobicity of the derivatives by the introduction of cyclohexyl group from CMCT.
We then examined the detection sensitivities of these uridine modications upon CMCT labelling. The limits of detection (LODs) dened as the amounts of the analytes at a signal-to-noise ratio (S/N) of 3 were employed to evaluate the detection sensitivities of uridine modications. The results demonstrated that the detection sensitivities of uridine modi-cations improved by 6 to 1408 fold upon CMCT labelling (Table 1). We reason that the introduction of positively charged quaternary ammonium group into uridine modications could greatly improve the ionization efficiency during ESI-MS analysis, which eventually leads to enhanced detection sensitivities. Upon CMCT labelling, the LODs of the CMCT labelled uridine   Table S1 in the ESI. † This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 1878-1891 | 1883 modications are at similar levels (Table 1). However, the LODs of these native uridine modications without CMCT labelling have relatively large differences (LODs ranging from 6.6 to 408.2 fmol, Table 1). Due to the large differences of the LODs among native uridine modications, the improved folds of detection sensitivities (LODs of native uridine modications/LODs of CMCT labelled uridine modications) were different among these uridine modications.
In addition to uridine modications, guanosine can also react with CMCT as in previous studies. 45,46 CMCT labelling can increase the detection sensitivity of guanosine by 7-fold, which is smaller than that for most of the uridine modications (Table 1). It can be seen that the ionization efficiency of guanosine is better than those of uridine modications and the increased detection sensitivity of guanosine by chemical labelling is therefore not as dramatic as that of uridine modications (Table 1). Although guanosine reacts with CMCT, this will not affect the sensitive detection of m 5 U and other uridine modications since they have distinctly different molecular weights (Table S1 in the ESI †).

Method validation
The calibration curves were constructed to quantitatively measure the modications in RNA. A mixture of uridine modication standards with a series of amounts ranging from 0.016 pmol to 16 pmol and xed amounts of isotope internal standards (D 3 -CMCT labelled derivatives) was prepared. The calibration curves were constructed by plotting the peak area ratios (analytes/IS) against the amounts of uridine modications with triplicate measurements. The results showed that good linearities were obtained with the coefficient of determination (R 2 ) being great than 0.9994 (Table S3 in the ESI †).
In addition, the accuracy of the established method was evaluated by comparing the measured contents of uridine modications to the theoretical contents of uridine modications. The intra-and inter-day relative standard deviations (RSDs) were calculated with different amounts of uridine modication standards for evaluation of the precision of the method. Three measurements over a day gave the intra-day RSDs, and the inter-day RSDs were determined by measuring samples for three consecutive days. The results showed that the intra-and inter-day RSDs for uridine modications were less than 15.3% and relative errors (REs) were less than 7.8% (Table   S4 in the ESI †), indicating that good precision and accuracy were achieved. Taken together, the results demonstrated that the developed method was reliable for the accurate quantication of uridine modications.

Determination of m 5 U in mRNA in mammalian cells
With the established method, we explored the potential existence of endogenous uridine modications in mRNA from cultured human cell lines. Obtaining highly pure mRNA is very important for the determination of the existence of new modi-cations in mRNA. In this respect, here we developed a method to purify mRNA by successive three different methods, including (1) small RNA (<200 nt) depletion, (2) PolyATtract mRNA isolation (twice), and (3) agarose gel electrophoresisbased purication (Fig. 5A and B).
As tRNA contains abundant m 5 U modication, 47 tRNA contamination could lead to a false positive result for the determination of m 5 U in mRNA. In this respect, small RNA (<200 nt) was rst depleted from total RNA using the E.Z.N.A. miRNA kit. The resulting RNA was further puried twice with the PolyATtract mRNA isolation system followed by agarose gel electrophoresis-based purication ( Fig. 5A and B). Four modi-cations (m 1 G, m 2 G, i 6 A, and t 6 A) that were reported to be only present in tRNA or rRNA were used to evaluate the purity of the isolated mRNA. 9,48,49 Shown in Fig. 5C, D and 5E are the extracted-ion chromatograms of the standards of these 4 This journal is © The Royal Society of Chemistry 2020 Chem. Sci., 2020, 11, 1878-1891 | 1885 modications, the detected modications from total RNA and from mRNA of HEK293T cells, respectively. The results demonstrated that all these 4 modications were clearly detected in total RNA (Fig. 5D), but were undetectable in mRNA (Fig. 5E), indicating the high purity of mRNA.
In addition, we used real-time quantitative PCR to evaluate the purity of the isolated mRNA. The detailed evaluation can be found in the ESI. † The real-time quantitative PCR analysis demonstrated that the potential contamination of small RNA was less than 0.023% in mRNA (Fig. S8 in the ESI †), i.e. less than 0.12 ng of small RNA in 500 ng of mRNA. We then analyzed m 5 U from 500 ng of mRNA. The peak of m 5 U from 500 ng of mRNA was clear and distinct (Fig. S9 in the ESI †). However, no signal of m 5 U was observed from 0.12 ng of small RNA (Fig. S9 in the ESI †), indicating that the m 5 U signal from 500 ng of mRNA indeed comes from mRNA, but not the potential trace level of small RNA. Similarly, the potential trace levels of 18S and 28S rRNA in mRNA should not compromise the detection of m 5 U in mRNA (details can be found in the ESI †). Moreover, the RNA-seq analysis demonstrated that 12 299 genes were detected in the isolated mRNA (Table S5 and S6 in the ESI †), indicating the good quality of the isolated mRNA.
We then analysed the potential uridine modications in mRNA. It turned out that among the 7 uridine modications (J, m 5 U, m 6 U, m 1 J, hm 5 U, mo 5 U, and mcm 5 U), only m 5 U and J were detectable in mRNA. Shown in Fig. 6A and B are the extracted-ion chromatograms of CMCT labelled m 5 U and J in mRNA, respectively, from various cultured human cells. Although CMCT labelled m 5 U, m 6 U, and m 1 J have the same molecular weight and the same MRM ion transition was used to monitor CMCT labelled m 5 U, m 6 U, and m 1 J (Table S1 in the ESI †), they could be well separated in HPLC (the retention times of m 5 U, m 6 U, and m 1 J were 21.06 min, 19.18 min and 18.55 min, respectively; Fig. S10A in the ESI †). Under these separation conditions, we conrmed that only m 5 U was detected in mRNA; no m 6 U or m 1 J was detected (Fig. S10B in the ESI †). Since there is no detectable m 6 U or m 1 J in mRNA of cultured human cells, we used the shorter gradient (Fig. 6) instead of the longer gradient (Fig. S10 in the ESI †) to detect m 5 U from mRNA.
It can be seen that m 5 U and J were undetectable in the control samples (only adding enzymes and omitting mRNA or in vitro transcribed RNA) (Fig. 6), which excludes the possibility that the detected m 5 U and J were from the contamination of enzymes used in mRNA digestion. To further conrm the detected m 5 U and J in mRNA, we also employed highresolution mass spectrometry to examine the CMCT labelled m 5 U and J. The results showed that the extracted-ion chromatograms, MS spectra and MS/MS spectra of CMCT/D 3 -CMCT labelled m 5 U and J from the cultured HEK293T cells are identical to those of the standards (Fig. 7 and S11 in the ESI †), further conrming the detected m 5 U and J in mRNA. These results collectively suggested that both m 5 U and J were distinctly present in mRNA of mammals.
J modication has been previously reported to be present in mRNA of mammals with a potential role in enhancing transcript stability. 35,36,50 It is worth noting that the universal existence of m 5 U in mRNA of various cell lines is rst reported in the current study. On the other hand, detection of m 5 U by LC-ESI-MS/MS without CMCT labelling showed that no m 5 U was observed (Fig. S12 in the ESI †), indicating that the direct analysis of m 5 U is challenging and our developed chemical labelling method is capable of detecting low abundance of m 5 U in mRNA.

Metabolic labelling of cells
To further conrm the occurrence of m 5 U in mRNA of mammals, we used stable isotope tracing monitored by mass spectrometry (Fig. 8A). It has been known that ATP and Lmethionine could be converted to S-adenosyl-L-methionine (SAM) by methionine adenosyltransferase, and SAM is a universal methylating agent for the methylation of nucleic acids. 51 We cultured human HEK293T cells in DMEM medium supplied with 0.3 mM stable isotope labelled L-methionine (D 3 -Met). Then mRNA was isolated and analysed by our developed method. The result showed D 3 -m 5 U was distinctly detected besides m 5 U (Fig. 8B). The concentration of unlabelled methionine in the DMEM medium is 0.2 mM. Thus, the percentage of D 3 -Met in medium is approximately 60% of the total methionine. And approximately half of the measured m 5 U carried the CD 3 group, which is comparable to the percentage of D 3 -Met in total methionine of the media. Extended culturing time of cells in the DMEM medium with added D 3 -Met will not increase the percentage of D 3 -m 5 U, further indicating that the methyl group of m 5 U is from SAM. Collectively, this result indicated that SAM was the methyl donor for the methylation of uridine in mRNA.

Content of m 5 U in different species of RNA
We then further quantitatively measured the content of m 5 U in different RNA species. In this respect, we isolated different types of RNA, including mRNA, 28S rRNA, 18S rRNA and small RNA (<200 nt) from HEK293T and HeLa cells. The puried 28S rRNA, 18S rRNA, and small RNA (<200 nt) were conrmed by agarose and polyacrylamide gel electrophoresis analysis (Fig. S13 in the ESI †).
Notably, m 5 U was detectable in all species of RNA in both HEK293T and HeLa cells. The measured contents of m 5 U in mRNA of various human cells and mouse tissues ranged from 0.001% to 0.0059% (m 5 U/U) ( Fig. 9A and B). In addition, the measured contents of J in mRNA ranged from 0.29% to 1.04% (J/U) (Fig. 9C and D), which are comparable to previously reported levels of J in mRNA of mammals, 13,36 indicating the good accuracy of the developed method.
The contents of m 5 U in small RNA (<200 nt) of HEK293T and HeLa cells were 1.9% and 1.6% (m 5 U/U), respectively (Fig. 9E). The contents of m 5 U in 28S and 18S rRNA of HEK293T and HeLa cells ranged from 0.0039% to 0.0057% (m 5 U/U) (Fig. 9E). These results showed that small RNA (<200 nt) contained the most abundant m 5 U, while mRNA and rRNA had similar levels of m 5 U. It was reported that m 5 U was an abundant modication in tRNA. 16 And a pervious study showed tRNA presents $90% of small RNA (<200 nt). 52 Thus, the detected m 5 U in small RNA should mainly come from tRNA.
It has been known that over 150 types of modications were identied in RNA, 53 but only a small number of modications were detected in mRNA of eukaryotes. 9,11 Here, we demonstrated the existence of appreciable levels of m 5 U (0.001% to 0.0059%, m 5 U/U) in mRNA of mammalian cells. The measured content of m 5 U in mRNA is comparable to that of recently identied m 6 Am ($0.003%, m 6 Am/A) and m 3 C ($0.001-0.005%, m 3 C/C) in mRNA of mammals. 22,54 It has been proposed that the modications of m 6 Am and m 3 C in mRNA may affect the mRNA stability and translation efficiency. 55,56 Although the potential role of m 5 U in mRNA remains elusive, we speculate that m 5 U in mRNA may affect its interaction with the ribosome as some other modications. m 5 U could also lead to the alteration of RNA secondary structures and recruit m 5 U-specic binding proteins, and eventually impact translation.
Evaluation of the level of m 5 U in mRNA upon siRNA knockdown or overexpression of TRMT2A In E. coli, TrmA catalyzes the formation of m 5 U in tRNA. 57 In Saccharomyces cerevisiae, Trm2p is responsible for the formation of m 5 U in tRNA. 58 TrmA and Trm2p catalyze methyl group transfer from S-adenosyl-methionine (SAM) to the C5 of U54 of tRNA. 59 In mammals, tRNA methyltransferase 2 homologs (TRMT2A and TRMT2B) are predicted to be the homologs of yeast Trm2p. 60 The predicted methyltransferase domain of TRMT2A has an identity of 36% with that of Trm2p. TRMT2A contains a RNA recognition motif (RRM) in the N-terminus. However, the shared identity between TRMT2B and Trm2p is 28%, and the TRMT2B protein does not have an RRM motif. 60 This indicates that TRMT2A is the likely mammalian homolog of Trm2p. 40 In this respect, here we examined whether TRMT2A was responsible for depositing m 5 U in mRNA upon siRNA knockdown or overexpression of the TRMT2A gene.
The real-time quantitative PCR and western blotting results demonstrated the efficient knockdown or overexpression of TRMT2A was achieved (Fig. S14 in the ESI †). Then we analyzed the level of m 5 U in small RNA and mRNA upon siRNA knockdown or overexpression of TRMT2A. The results showed a signicant decrease of m 5 U in small RNA (p ¼ 2.0 Â 10 À5 , Fig. 10A), indicating that TRMT2A may be responsible for the formation of m 5 U in small RNA. However, no obvious increase of m 5 U in small RNA was observed for the overexpression of TRMT2A (p ¼ 0.085, Fig. 10A). We reason that the increased amount of TRMT2A does not affect the homeostasis of m 5 U in small RNA in cells, which was also reported by a recent study. 40 The rescue experiment showed the certain recovery of m 5 U in small RNA (Fig. 10A). As for the m 5 U in mRNA, we observed a signicant decrease (p ¼ 0.027) and signicant increase (p ¼ 6.3 Â 10 À5 ) of m 5 U in mRNA upon siRNA knockdown and overexpression of TRMT2A, respectively (Fig. 10B). The rescue experiment showed the efficient recovery of m 5 U in mRNA (Fig. 10B). The results demonstrated that TRMT2A should be responsible for the formation of m 5 U in mRNA.
Deciphering the distribution and dynamic change of m 5 U in mRNA will uncover the underlying mechanism of m 5 U. The transcriptome-wide mapping of m 5 U could be achieved by affinity pulldown of the m 5 U-containing RNA fragment followed by high-throughput sequencing. In addition, some modications in RNA can lead to reverse transcription arrest or misincorporation of dNTP, which has been utilized as a characteristic signature for locating modications by subsequent sequencing analysis. 61 By virtue of this strategy of reverse transcription induced arrest or misincorporation of dNTP, people may evolve enzymes that are suitable for probing m 5 U and develop method for mapping m 5 U at single-base resolution. Moreover, the exploration of the m 5 U-specic binding proteins may reveal the readers of m 5 U and promote the understanding of the functional roles of m 5 U.

Conclusions
In summary, we established a method of CMCT labelling coupled with LC-ESI-MS/MS analysis for the sensitive determination of uridine modications in RNA. The introduction of positively charged quaternary ammonium from CMCT can greatly increase the detection sensitivities of uridine modications. Using the developed method, we identied the universal existence of m 5 U in mRNA of cultured mammalian cells and tissues. In addition, the stable isotope tracing monitored by mass spectrometry revealed that the methylation group of m 5 U was from SAM. The identication of demethylases and reader proteins of m 5 U will facilitate understanding the functions of m 5 U in the future.

Data availability
Gene expression by RNA-seq is available in the Gene Expression Omnibus (GEO) database at NCBI with accession number GSE135049.

Conflicts of interest
The authors declare no competing nancial interest.