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. Author manuscript; available in PMC: 2023 Jul 1.
Published in final edited form as: Methods. 2021 May 18;203:1–4. doi: 10.1016/j.ymeth.2021.05.010

Pseudouridine RNA modification detection and quantification by RT-PCR

Wen Zhang a, Tao Pan b,*
PMCID: PMC8599501  NIHMSID: NIHMS1709356  PMID: 34020036

Abstract

Pseudourine (Ψ) is the most abundant cellular RNA modification, present in tRNA, rRNA, snRNA, mRNA, long noncoding RNA (lncRNA), and others. Ψ sites and fractions are dynamically regulated in stress response and across development stages. Although high throughput Ψ sequencing methods based on N-Cyclohexyl-N’-(2-morpholinoethyl)carbodiimide (CMC) reaction are available for Ψ detection transcriptome-wide, a simple method for the analysis of specific, targeted Ψ sites and their fraction quantitation is needed to better investigate Ψ function. Here, we describe an RT-PCR and gel electrophoresis based method that can sensitively and quantitatively assess Ψ at single-nucleotide resolution in mRNA/lncRNA, termed CMC-RT and ligation assisted PCR analysis of Ψ modification (CLAP). The principle of the CMC-method is the reverse transcription stop induced by the CMC-Ψ adduct. In CLAP, CMC reaction is first carried out with the RNA sample. Reverse transcription using a non-processive RT produces two cDNA products for each RNA transcript, one with the 3’ end at the Ψ site, the other read-through product from the unmodified RNA. Using splint oligonucleotide assisted site-specific ligation, these two cDNA products are then visualized on a gel as two distinct PCR products in the same lane corresponding to the Ψ-modified and unmodified target site. CLAP validates Ψ sites identified by high throughput sequencing, quantifies Ψ levels in mRNA and lncRNA, and enables convenient and rapid investigation on the function and mechanism of the Ψ modification.

Keywords: pseudouridine, CMC, mRNA, lncRNA, RT-PCR

1. Introduction

Pseudourine (Ψ) is the most abundant modification in cellular RNA [1]. Several CMC based next-generation sequencing methods identified Ψ in mRNA and long noncoding RNA (lncRNA) [2–5]. Ψ is generated through the isomerization of uridine catalyzed by pseudouridine synthases (PUS). Ψ has an additional hydrogen bond donor at N1 position (Fig. 1A) and more rotational freedom due to the C1’-C5 carbon-carbon bond compared to unmodified uridine. Ψ can form hydrogen bond to the ribophosphate backbone to rigidify RNA structure and improve base stacking [1]. In tRNA, Ψ in T and anticodon stem loops can increase tRNA stability by maintaining the regional structure. Ψ in the anticodon stem loop can influence translation efficiency and accuracy by enhancing codon-anticodon base-pairing [1, 6]. Ψ-containing tRNA fragments regulate protein synthesis in stem cells by targeting the translation initiation complex [7]. In rRNA, Ψ plays roles in rRNA processing and ribosome assembly. Specific Ψ modifications in the rRNA decoding center, peptidyl transferase center, and A-site finger are crucial for rRNA processing, ribosome-tRNA binding and protein synthesis fidelity [1, 6, 8]. In snRNA, Ψ is crucial for snRNP biogenesis and pre-mRNA splicing [9, 10]. A Ψ modification in U2 snRNA helps maintain the bulged branch-point structure which is crucial for pre-mRNA splicing [6, 8]. In mammalian mRNA, Ψ is one of the major RNA modifications, at a global level only several-folds below that of the N6-methyladenosine (m6A) modification measured by quantitative LC/MS/MS [5]. Additional Ψ modifications can be induced in mRNA by stress [2, 4, 5], suggesting dynamic regulation for biological regulation. Ψ modification in stop codons enables efficient stop codon read-through [11]. Ψ in mRNA coding sequences can alter protein synthesis speed as well as codon usage [12]. mRNA structure can modulate pseudouridinylation by the PUS1 enzyme, implicating an RNA structure dependent mechanism of mRNA pseudouridylation [13].

Fig. 1.

Fig. 1.

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Chemical structure of Ψ, CMC-Ψ adduct, and schematics of CLAP. (A-B) Chemical structure of Ψ (A), CMC-Ψ adduct (B). Modified atoms are in red. CMC is in bright blue. Ψ has an extra hydrogen bond donor comparing to unmodified U. (C) Schematics of CLAP. CLAP starts with ± CMC treatment. 5mer RNA (RNA-5 blocker) is ligated to free 5′ end of RNA to disrupt splint ligation and reduce background from undesired RNA fragmentation in CMC reaction. RNA is shown as a dark grey line, RNA-5 as black line, cDNA as a green line, adapter oligo as a red line, splint ligation oligo as a bright blue line, the forward and reverse PCR primers as red and green arrows, respectively, and the primer binding site in Ψ +CMC and −CMC as the red line at the end,

A sensitive and quantitative Ψ analysis method would improve our ability to investigate Ψ function in low abundance mRNA and lncRNA. In mRNA/lncRNA, Ψ at each site is always sub-stoichiometric, making the determination of its modification fraction a crucial parameter in understanding its biological function. Ψ can be directly detected and quantified by thin-layer chromatography based methods [5, 14, 15], but, these methods only work well with abundant cellular RNAs. For mRNA/lncRNA, current high-throughput Ψ sequencing methods all utilize the reaction of N-cyclohexyl-N’-(2-morpholinoethyl)carbodiimide (CMC) with Ψ (Fig. 1B)[16]. CMC reacts with Ψ to form a CMC-Ψ adduct on the Watson-Crick face that interferes with reverse transcription at the Ψ sites, leaving a stop or mutation signature in the cDNA product in high throughput sequencing [2–4, 17]. Although powerful, sequencing-based method cannot be routinely used, and the current sequencing strategies do not reliably provide quantitative information on Ψ fraction at specific site of interest. For specific, targeted Ψ sites, the CMC-Ψ adduct can be detected by primer extension and denaturing gel analysis which was particularly useful for Ψ sites in abundant rRNA and snRNA. For low abundance mRNA/lncRNAs, however, PCR must be incorporated for signal amplification, for example as described in reference [18]. The challenge of incorporating PCR is to not only enable Ψ detection, but also maintain quantitative information of Ψ modification fraction.

Here, we describe a method of Ψ analysis derived from CMC reaction based RT-PCR and gel electrophoresis (Fig. 1C). The method, termed CMC-RT and Ligation Assisted PCR analysis of Ψ modification (CLAP), measures the RT stop induced by the CMC-Ψ adduct and its corresponding read-through cDNA derived from unmodified RNA transcript of the same sequence. Through selective ligation of an oligonucleotide to the CMC-Ψ stopped cDNA, both cDNA products are PCR amplified in the same tube using the same primers. The Ψ modification fraction is readily determined by quantifying the two product bands in the same lane. We have shown that CLAP is sensitive and quantitative in analyzing Ψ sites in mRNA/lncRNA [19].

2. Methods

2.1. CMC treatment of RNA samples

The CLAP method relies on the CMC-Ψ adduct formed through a series of chemical treatment of the RNA sample. Discovered in 1970s [20], CMC reacts with U- and G-like residues; it has been widely used in Ψ studies by primer extension and gel electrophoresis [16], and high-throughput sequencing [2–5]. Under urea denaturing conditions at pH 8.3, CMC reacts with G, U, Ψ, and inosine in RNA to form N3-CMC and N1-CMC adducts. All except the N3-CMC adduct in Ψ are removed under basic conditions at pH 10.4. The surviving CMC-Ψ adduct contains a bulky group on the Watson-Crick face of the Ψ base which stops reverse transcription one nucleotide 3’ to the target Ψ site using a non-processive RT such as the AMV RT. This section describes the CMC reaction of the RNA sample. Total RNA was first treated with freshly made CMC. An alkaline treatment of the CMC reacted RNA is then applied to reverse the non-Ψ derived CMC adducts on the RNA.

  1. Use two tubes of total RNA in 12 μl with the mass ratio of 1.5:1 (e.g. 15 μg : 10 μg) are mixed with 24 μl TEU buffer (50 mM Tris-HCl, pH 8.3, 4 mM EDTA, 7 M urea) for CMC and mock treatment, respectively.

  2. Add 4 μl 1 M CMC freshly dissolved in TEU buffer (+) or 4 μl TEU buffer (−) to each tube for +CMC or −CMC mock reaction for a final condition of 0.7× TEU buffer and 0.1 M CMC in 40 μl.

  3. Incubate samples at 30°C for 16 hours after mixing.

  4. Dilute the reaction mixture to 200 μl with 160 μl crush-soak buffer (50 mM KOAc, 200 mM KCl, pH 7). Add 1 μl 5 μg/μl glycogen. Recover RNA by adding 2.7× volume (~540 μl) of ethanol and incubation at −80°C for at least 1 hour.

  5. Pellet RNA by centrifuging the samples at 17,000g for 30 min at 4°C.

  6. Wash RNA pellet with 500 μl of 70% ethanol twice to remove excess CMC. For each wash, resuspend RNA, incubated at −80°C, and pellet by centrifugation.

  7. Resuspend RNA pellet with 40 μl reverse buffer (50 mM Na2CO3, 2 mM EDTA, pH 10.4), incubate the mixture at 37 °C for 6 hours.

  8. Dilute the reaction mixture to 200 μl with 160 μl crush-soak buffer and repeat ethanol precipitation as in steps 4–6.

  9. Resuspend RNA in sterile water. If needed, measure RNA concentration by Nanodrop.

2.2. RNA 5’phosphorylation and blocker ligation

The CMC reaction and the reversal steps can cause RNA fragmentation, resulting in elevated background signals and false-positive results. In order to reduce background signal at target sites, a “blocking” RNA ligation step is added in our method. The ligated 5-mer RNA oligonucleotide interrupts the splint ligation by fragmented RNA at target Ψ site and reduces background. Ligation of this blocking 5-mer RNA requires a 5’ phosphate of the CMC-induced fragment; therefore 5’ phosphorylation is first carried out before ligation.

  1. To 6 μg +CMC treated or 4 μg −CMC treated total RNA in 6.5 μl, add 1μl 10X T4 PNK reaction buffer (B0201S, NEB), 1 μl 1 mM ATP, 0.5 μl RNase inhibitor (M0307L, NEB), and 1 μl 10 U/μl T4 PNK (M0201L, NEB) to each tube and mix.

  2. Incubate samples at 37°C for 30 min, followed by a quick spin.

  3. Add 1 μl 10×T4 RNA Ligase Reaction Buffer (B0216L, NEB), 1 μl 100 μM 5’ RNA blocker oligo (/5AmMC6/rArCrCrCrA, IDT), 1 μl 1 mM ATP, 1 μl RNase inhibitor, 3 μl DMSO, 2 μl sterile water, and 1 μl 10 U/μl T4 RNA ligase I (M0204L, NEB).

  4. Incubate the reaction mixtures at 16°C for 16 hours.

  5. Add 1.2 μl 200 mM EDTA to each tube to stop the reaction.

2.3. Reverse transcription and splint ligation

It is well established that the AMV reverse transcriptase stops one nucleotide 3’ to the target CMC-Ψ adduct site. Reverse transcription using this RT generates two cDNA products, a shorter one from the Ψ-modified transcript, and a longer one from the unmodified transcript of the same sequence. In order to amplify the short and long cDNA products using just one pair of primers which more accurately maintains the quantitative information of Ψ level, an adaptor oligonucleotide is ligated only to the short cDNA product guided by a splint oligonucleotide. This adaptor has the same primer binding site as the long cDNA product which enables simultaneous amplification of both in the same PCR reaction.

  1. Use 3 μl ±CMC ligation mixture from section 2.2 for RT with AMV reverse transcriptase (M0277L, NEB). To this mixture, add 1 μl 10× annealing buffer (250 mM Tris-HCl, 480 mM KCl, pH 7.4) and 1 μl 0.5 μM target-specific RT primer (~3:1 ratio of primer:RNA).

  2. Incubate the mixture at 93°C for 2 min followed by incubation at room temperature for 3 min to anneal the primer to the RNA.

  3. Add 5 μl of 2× AMV RT reaction mixture (1.2 U/μl AMV RT, 2 × AMV RT buffer, 1 mM of each dNTP) to each tube for a final condition of 0.6 U/μl AMV RT, 1× AMV RT buffer, and 0.5 mM of each dNTP. Incubate the mixture at 42°C for 1 hour.

  4. Inactivate AMV RT by incubating the mixture at 85 °C for 5 min followed by putting on ice immediately.

  5. Add 1 μl 5 U/μl RNase H (NEB, M0297L) to each tube and incubate at 37 °C for 20 min to digest the RNA complementary to cDNA.

  6. Inactivate RNase H by incubation at 85 °C for 5 min followed by putting on ice immediately.

  7. Add 1 μl 3’-adaptor/Splint oligo mix (1.5 μM adaptor oligonucleotides and 1.5 μM splint oligonucleotides) to the above RT mixture. Incubate the mixture at 75 °C for 3 min followed by incubation at room temperature for 3 min to anneal the adaptor and splint oligonucleotides.

  8. Add 4 μl of 4× ligation mixture (40 U/μl of T4 DNA ligase (NEB, M0202L), 4× T4 DNA ligase reaction buffer, 50% DMSO) to a final concentration of 10 U/μl of T4 DNA ligase, 1× T4 DNA ligase reaction buffer, and 12.5% DMSO to ligate the adaptor oligonucleotides to the 3’ end of the truncated cDNA.

  9. Incubate the mixture at 16°C for 16 h.

  10. Heat the mixture at 65 °C for 10 min followed by putting on ice immediately to deactivate the T4 DNA ligase.

2.4. PCR and gel electrophoresis

The site-specific splint ligation enables PCR amplification of the short and long cDNA product with only one pair of primer. Both primers of each pair should have the same or similar melting temperatures to enable successful PCR reaction. The size difference of the two PCR products is around 30 nucleotides to allow for optimal separation using 10% native PAGE gel electrophoresis. After staining with SYBR gold, the two bands can be visualized and quantified with Bio-Rad ChemiDoc imaging system.

  1. Use 2 μl of the ligation mixture from section 2.3 for PCR reactions using NEB Q5 High-Fidelity DNA Polymerase (M0491L, NEB) in 35 μl.

  2. Add 3.5 μl 5 μM forward primer, 3.5 μl 5 μM reverse primer, and 2 μl cDNA synthesis mixture from above to each PCR tube.

  3. Add 26 μl 1.35×Q5 DNA polymerase mixture to each tube for a final condition of 1× Q5 reaction buffer, 1× Q5 high GC enhancer (B9027S, NEB), 200 μM of each dNTP, 0.5 μM forward and reverse primers, and 0.02 U/μl Q5 high-fidelity DNA polymerase.

  4. Do PCR at suggested PCR conditions with specific annealing temperature and PCR cycles for each site.

  5. Mix half of the PCR reaction mixture (17.5 μl) with 3.5 μl of 6×TriTrack DNA Loading Dye (ThermoFisher, R1161).

  6. Load the mixture to a pre-run 10% native PAGE gel containing 1× TBE, together with low range DNA ladder (SM1193, Thermo) for the identification of the target bands.

  7. Stain the gel with SYBR gold nucleic acid gel stain (S11494, Thermo) for 10 min in 1× TBE. Scan the gel using Bio-Rad ChemiDoc imaging system and quantify the target bands using Image Lab (Bio-Rad).

In each lane, the lower band represents the Ψ modified RNA and higher band the unmodified RNA of the same sequence (Fig. 2).

Fig. 2.

Fig. 2.

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Representative CLAP gel image of target Ψ sites in HeLa and MCF7 cell lines. (A) Ψ519 in mRNA, eEF1A1. (B) Ψ5590 in lncRNA, MALAT1. Lower bands represent Ψ modified RNA and higher band represent unmodified RNA. Ψ modification fraction can be obtained by quantifying the two bands in each lane.

3. Results and discussion

We have developed a sensitive and quantitative method for site-specific Ψ analysis in low abundance mRNA and lncRNA. The method has been successfully applied to the quantification of Ψ in rRNA, mRNA and lncRNA [19]. In that published work, using model Ψ and unmodified oligonucleotides we established the quantitative feature of the CLAP method. We validated the quantitative nature of 3 rRNA Ψ sites by PCR-based CLAP and gel-based primer extension without amplification. We validated and measured the modification fractions of target Ψ sites identified in high-throughput sequencing [5] in a high abundance mRNA (eEF1A1), a lncRNA (MALAT1), and low abundance mRNAs (HPRT1, PSME2) in HEK293T, HeLa, and MCF7. We found similar levels of Ψ modification fractions at these sites in these 3 cell lines, and the Ψ levels at these sites ranged from 30–80%. We also showed comparative Ψ levels measured with either total RNA or polyA-selected RNA for the low abundance mRNA sites.

In conclusion, CLAP provides a simple and radioactivity-free approach for Ψ analysis for investigation and characterization of Ψ sites in mRNA/lncRNA. Since Ψ is dynamically regulated and responds to stress, the ability to rapidly track the modification fraction change will be a big plus to help understand the context-dependent function of Ψ modification.

Highlights.

  • We describe a method to analyze pseudouridine (Ψ) modifications in mRNA and lncRNA.

  • The RT-PCR method validates Ψ sites from sequencing or other approaches.

  • The RT-PCR method quantifies the modification level of Ψ sites.

Acknowledgments

This work was supported by the National Institutes of Health (RM1HG008935 to T.P.)

Footnotes

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The authors declare no conflict of interest for the article: Pseudouridine RNA modification detection and quantification by RT-PCR.

References

  • [1].Charette M, Gray MW, Pseudouridine in RNA: what, where, how, and why, IUBMB Life 49(5) (2000) 341–51. [DOI] [PubMed] [Google Scholar]
  • [2].Carlile TM, Rojas-Duran MF, Zinshteyn B, Shin H, Bartoli KM, Gilbert WV, Pseudouridine profiling reveals regulated mRNA pseudouridylation in yeast and human cells, Nature 515(7525) (2014) 143–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [3].Lovejoy AF, Riordan DP, Brown PO, Transcriptome-wide mapping of pseudouridines: pseudouridine synthases modify specific mRNAs in S. cerevisiae, PLoS One 9(10) (2014) e110799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [4].Schwartz S, Bernstein DA, Mumbach MR, Jovanovic M, Herbst RH, Leon-Ricardo BX, Engreitz JM, Guttman M, Satija R, Lander ES, Fink G, Regev A, Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA, Cell 159(1) (2014) 148–162. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [5].Li X, Zhu P, Ma S, Song J, Bai J, Sun F, Yi C, Chemical pulldown reveals dynamic pseudouridylation of the mammalian transcriptome, Nat Chem Biol 11(8) (2015) 592–7. [DOI] [PubMed] [Google Scholar]
  • [6].Li X, Ma S, Yi C, Pseudouridine: the fifth RNA nucleotide with renewed interests, Curr Opin Chem Biol 33 (2016) 108–16. [DOI] [PubMed] [Google Scholar]
  • [7].Guzzi N, Ciesla M, Ngoc PCT, Lang S, Arora S, Dimitriou M, Pimkova K, Sommarin MNE, Munita R, Lubas M, Lim Y, Okuyama K, Soneji S, Karlsson G, Hansson J, Jonsson G, Lund AH, Sigvardsson M, Hellstrom-Lindberg E, Hsieh AC, Bellodi C, Pseudouridylation of tRNA-Derived Fragments Steers Translational Control in Stem Cells, Cell 173(5) (2018) 1204–1216 e26. [DOI] [PubMed] [Google Scholar]
  • [8].Ge J, Yu YT, RNA pseudouridylation: new insights into an old modification, Trends Biochem Sci 38(4) (2013) 210–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [9].Wu G, Xiao M, Yang C, Yu YT, U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP, EMBO J 30(1) (2011) 79–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [10].Wu G, Adachi H, Ge J, Stephenson D, Query CC, Yu YT, Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly, EMBO J 35(6) (2016) 654–67. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [11].Karijolich J, Yu YT, Converting nonsense codons into sense codons by targeted pseudouridylation, Nature 474(7351) (2011) 395–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [12].Eyler DE, Franco MK, Batool Z, Wu MZ, Dubuke ML, Dobosz-Bartoszek M, Jones JD, Polikanov YS, Roy B, Koutmou KS, Pseudouridinylation of mRNA coding sequences alters translation, Proc Natl Acad Sci U S A 116(46) (2019) 23068–23074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [13].Carlile TM, Martinez NM, Schaening C, Su A, Bell TA, Zinshteyn B, Gilbert WV, mRNA structure determines modification by pseudouridine synthase 1, Nat Chem Biol 15(10) (2019) 966–974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [14].Zhao X, Yu YT, Detection and quantitation of RNA base modifications, RNA 10(6) (2004) 996–1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [15].Liu N, Parisien M, Dai Q, Zheng G, He C, Pan T, Probing N6-methyladenosine RNA modification status at single nucleotide resolution in mRNA and long noncoding RNA, RNA 19(12) (2013) 1848–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [16].Bakin A, Ofengand J, Four newly located pseudouridylate residues in Escherichia coli 23S ribosomal RNA are all at the peptidyltransferase center: analysis by the application of a new sequencing technique, Biochemistry 32(37) (1993) 9754–62. [DOI] [PubMed] [Google Scholar]
  • [17].Zhou KI, Clark WC, Pan DW, Eckwahl MJ, Dai Q, Pan T, Pseudouridines have context-dependent mutation and stop rates in high-throughput sequencing, RNA Biol 15(7) (2018) 892–900. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [18].Lei Z, Yi C, A Radiolabeling-Free, qPCR-Based Method for Locus-Specific Pseudouridine Detection, Angew Chem Int Ed Engl 56(47) (2017) 14878–14882. [DOI] [PubMed] [Google Scholar]
  • [19].Zhang W, Eckwahl MJ, Zhou KI, Pan T, Sensitive and quantitative probing of pseudouridine modification in mRNA and long noncoding RNA, RNA 25(9) (2019) 1218–1225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • [20].Ho NW, Gilham PT, Reaction of pseudouridine and inosine with N-cyclohexyl-N’-beta-(4-methylmorpholinium)ethylcarbodiimide, Biochemistry 10(20) (1971) 3651–7. [PubMed] [Google Scholar]

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