Skip to main content
See More

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

A Ψ–Ψ codon–anticodon pairing in nonsense suppression and translational recoding

Nature Chemical Biology (2025)Cite this article

Subjects

Abstract

Pseudouridine (Ψ) is known for decades but its flexibility in base pairing remains unclear. This study engineers artificial box H/ACA guide RNAs to direct pseudouridylation at the uridine of a premature termination codon (PTC; UAA, UAG or UGA) within an intronless mRNA and U36 of the anticodon of a matching tRNA in yeast and human cells. Targeted pseudouridylation leads to the formation of a Ψ–Ψ codon–anticodon pair, which, together with the other two Watson–Crick base pairs in the codon–anticodon duplex, greatly improves codon–anticodon recognition, robustly promoting PTC readthrough. The intronless mRNA level remains unchanged with or without guide RNAs. Additionally, pseudouridylation does not impact tRNA stability or charging. Our results show that nonsense suppression is promoted by the high affinity of the Ψ–Ψ pair, which is verified by melting curve analysis. This work identifies an unusual Ψ–Ψ base pair, which contributes greatly to codon–anticodon recognition and translational recoding.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Box H/ACA gRNA-directed PTC (mRNA) pseudouridylation.
Fig. 2: Box H/ACA gRNA-directed tRNA pseudouridylation.
Fig. 3: Quantitative analysis of targeted pseudouridylation.
Fig. 4: cup1 nonsense suppression assay verifying the Ψ–Ψ codon–anticodon pair.
Fig. 5: Ψ–Ψ-mediated nonsense suppression using the mCherry reporter.
Fig. 6: Nonsense suppression assay verifying the Ψ–Ψ codon–anticodon pair in the mCherry gene context in human cells.

Similar content being viewed by others

Data availability

The reference genome of S. cerevisiae R64-3-1 (GCF_000146045) and mCherry coding sequence (AY678264) were retrieved from the National Center for Biotechnology Information (NCBI). The sequence data generated in this study were deposited to the NCBI Gene Expression Omnibus under accession code GSE299290. Source data are provided with this paper.

References

  1. Ishigaki, Y., Li, X., Serin, G. & Maquat, L. E. Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell 106, 607–617 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Isken, O. & Maquat, L. E. The multiple lives of NMD factors: balancing roles in gene and genome regulation. Nat. Rev. Genet. 9, 699–712 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Brogna, S., McLeod, T. & Petric, M. The meaning of NMD: translate or perish. Trends Genet. 32, 395–407 (2016).

    Article  CAS  PubMed  Google Scholar 

  4. Morais, P., Adachi, H. & Yu, Y. T. Suppression of nonsense mutations by new emerging technologies. Int. J. Mol. Sci. 21, 4394 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Adachi, H. et al. Targeted pseudouridylation: an approach for suppressing nonsense mutations in disease genes. Mol. Cell 83, 637–651.e9 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Karijolich, J. & Yu, Y. T. Converting nonsense codons into sense codons by targeted pseudouridylation. Nature 474, 395–398 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Song, J. et al. CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons. Mol. Cell 83, 139–155.e9 (2023).

    Article  CAS  PubMed  Google Scholar 

  8. Charette, M. & Gray, M. W. Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49, 341–351 (2000).

    Article  CAS  PubMed  Google Scholar 

  9. Davis, D. R. Stabilization of RNA stacking by pseudouridine. Nucleic Acids Res. 23, 5020–5026 (1995).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Dai, Q. et al. Quantitative sequencing using BID-seq uncovers abundant pseudouridines in mammalian mRNA at base resolution. Nat. Biotechnol. 41, 344–354 (2023).

    Article  CAS  PubMed  Google Scholar 

  11. Xiao, M., Yang, C., Schattner, P. & Yu, Y. T. Functionality and substrate specificity of human box H/ACA guide RNAs. RNA 15, 176–186 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Huang, C., Wu, G. & Yu, Y. T. Inducing nonsense suppression by targeted pseudouridylation. Nat. Protoc. 7, 789–800 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Song, J., Zhuang, Y. & Yi, C. Programmable RNA base editing via targeted modifications. Nat. Chem. Biol. 20, 277–290 (2024).

    Article  CAS  PubMed  Google Scholar 

  14. Ganot, P., Bortolin, M. L. & Kiss, T. Site-specific pseudouridine formation in preribosomal RNA is guided by small nucleolar RNAs. Cell 89, 799–809 (1997).

    Article  CAS  PubMed  Google Scholar 

  15. Ni, J., Tien, A. L. & Fournier, M. J. Small nucleolar RNAs direct site-specific synthesis of pseudouridine in ribosomal RNA. Cell 89, 565–573 (1997).

    Article  CAS  PubMed  Google Scholar 

  16. Yu, Y. T. & Meier, U. T. RNA-guided isomerization of uridine to pseudouridine—pseudouridylation. RNA Biol. 11, 1483–1494 (2014).

    Article  PubMed  Google Scholar 

  17. Meier, U. T. & Blobel, G. NAP57, a mammalian nucleolar protein with a putative homolog in yeast and bacteria. J. Cell Biol. 127, 1505–1514 (1994).

    Article  CAS  PubMed  Google Scholar 

  18. Chen, C., Zhao, X., Kierzek, R. & Yu, Y. T. A flexible RNA backbone within the polypyrimidine tract is required for U2AF65 binding and pre-mRNA splicing in vivo. Mol. Cell. Biol. 30, 4108–4119 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Adachi, H. & Yu, Y. T. Pseudouridine-mediated stop codon readthrough in S. cerevisiae is sequence context-independent. RNA 26, 1247–1256 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Fernandez, I. S. et al. Unusual base pairing during the decoding of a stop codon by the ribosome. Nature 500, 107–110 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Roy, B., Leszyk, J. D., Mangus, D. A. & Jacobson, A. Nonsense suppression by near-cognate tRNAs employs alternative base pairing at codon positions 1 and 3. Proc. Natl Acad. Sci. USA 112, 3038–3043 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Blanchet, S., Cornu, D., Argentini, M. & Namy, O. New insights into the incorporation of natural suppressor tRNAs at stop codons in Saccharomyces cerevisiae. Nucleic Acids Res. 42, 10061–10072 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Blanchet, S. et al. Deciphering the reading of the genetic code by near-cognate tRNA. Proc. Natl Acad. Sci. USA 115, 3018–3023 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Luo, N. et al. Near-cognate tRNAs increase the efficiency and precision of pseudouridine-mediated readthrough of premature termination codons. Nat. Biotechnol. 43, 114–123 (2025).

    Article  CAS  PubMed  Google Scholar 

  25. Hofhuis, J. et al. The functional readthrough extension of malate dehydrogenase reveals a modification of the genetic code. Open Biol. 6, 160246 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  26. Potapov, V. et al. Base modifications affecting RNA polymerase and reverse transcriptase fidelity. Nucleic Acids Res. 46, 5753–5763 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Chen, T. H., Potapov, V., Dai, N., Ong, J. L. & Roy, B. N1-methyl-pseudouridine is incorporated with higher fidelity than pseudouridine in synthetic RNAs. Sci. Rep. 12, 13017 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Kierzek, E. et al. The contribution of pseudouridine to stabilities and structure of RNAs. Nucleic Acids Res. 42, 3492–3501 (2014).

    Article  CAS  PubMed  Google Scholar 

  29. Rintala-Dempsey, A. C. & Kothe, U. Eukaryotic stand-alone pseudouridine synthases—RNA modifying enzymes and emerging regulators of gene expression? RNA Biol. 14, 1185–1196 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Borchardt, E. K., Martinez, N. M. & Gilbert, W. V. Regulation and function of RNA pseudouridylation in human cells. Annu. Rev. Genet. 54, 309–336 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lesser, C. F. & Guthrie, C. Mutational analysis of pre-mRNA splicing in Saccharomyces cerevisiae using a sensitive new reporter gene, CUP1. Genetics 133, 851–863 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. 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, 9754–9762 (1993).

    Article  CAS  PubMed  Google Scholar 

  33. Huang, C., Karijolich, J. & Yu, Y. T. Detection and quantification of RNA 2′-O-methylation and pseudouridylation. Methods 103, 68–76 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Grosjean, H., Keith, G. & Droogmans, L. Detection and quantification of modified nucleotides in RNA using thin-layer chromatography. Methods Mol. Biol. 265, 357–391 (2004).

    CAS  PubMed  Google Scholar 

  35. Zhao, X. & Yu, Y. T. Detection and quantitation of RNA base modifications. RNA 10, 996–1002 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Ogden, R. C., Lee, M. C. & Knapp, G. Transfer RNA splicing in Saccharomyces cerevisiae: defining the substrates. Nucleic Acids Res. 12, 9367–9382 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. del Rey, F. J., Donahue, T. F. & Fink, G. R. sigma, a repetitive element found adjacent to tRNA genes of yeast. Proc. Natl Acad. Sci. USA 79, 4138–4142 (1982).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Keith, G. The primary structures of two arginine tRNAs (anticodons C-C-U and mcm5a2U-C-ψ) and of glutamine tRNA (anticodon C-U-G) from bovine liver. Nucleic Acids Res. 12, 2543–2547 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Song, J. et al. Differential roles of human PUS10 in miRNA processing and tRNA pseudouridylation. Nat. Chem. Biol. 16, 160–169 (2020).

    Article  CAS  PubMed  Google Scholar 

  40. Dunin-Horkawicz, S. et al. MODOMICS: a database of RNA modification pathways. Nucleic Acids Res. 34, D145–D149 (2006).

    Article  CAS  PubMed  Google Scholar 

  41. Xu, H. et al. Absolute quantitative and base-resolution sequencing reveals comprehensive landscape of pseudouridine across the human transcriptome. Nat. Methods 21, 2024–2033 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Nir, R. et al. A systematic dissection of determinants and consequences of snoRNA-guided pseudouridylation of human mRNA. Nucleic Acids Res. 50, 4900–4916 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Schwartz, S. et al. Transcriptome-wide mapping reveals widespread dynamic-regulated pseudouridylation of ncRNA and mRNA. Cell 159, 148–162 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Ma, X. et al. Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism. EMBO J. 24, 2403–2413 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Vitali, P. & Kiss, T. Cooperative 2′-O-methylation of the wobble cytidine of human elongator tRNAMet(CAT) by a nucleolar and a Cajal body-specific box C/D RNP. Genes Dev. 33, 741–746 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Joardar, A., Malliahgari, S. R., Skariah, G. & Gupta, R. 2′-O-methylation of the wobble residue of elongator pre-tRNAMet in Haloferax volcanii is guided by a box C/D RNA containing unique features. RNA Biol. 8, 782–791 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Zhang, M. et al. A snoRNA–tRNA modification network governs codon-biased cellular states. Proc. Natl Acad. Sci. USA 120, e2312126120 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Rozov, A., Demeshkina, N., Westhof, E., Yusupov, M. & Yusupova, G. New structural insights into translational miscoding. Trends Biochem. Sci. 41, 798–814 (2016).

    Article  CAS  PubMed  Google Scholar 

  49. Demeshkina, N., Jenner, L., Westhof, E., Yusupov, M. & Yusupova, G. New structural insights into the decoding mechanism: translation infidelity via a G•U pair with Watson–Crick geometry. FEBS Lett. 587, 1848–1857 (2013).

    Article  CAS  PubMed  Google Scholar 

  50. Hoernes, T. P. et al. Translation of non-standard codon nucleotides reveals minimal requirements for codon–anticodon interactions. Nat. Commun. 9, 4865 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Koiwai, O. & Miyazaki, M. The primary structure of non-initiator methionine transfer ribonucleic acid from Bakers’ yeast. II. Partial digestion with ribonuclease T1 and derivation of the complete sequence. J. Biochem. 80, 951–959 (1976).

    Article  CAS  PubMed  Google Scholar 

  52. Harada, F., Matsubara, M. & Kato, N. Stable tRNA precursors in HeLa cells. Nucleic Acids Res. 12, 9263–9269 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Piper, P. W. The nucleotide sequence of a methionine tRNA which functions in protein elongation in mouse myeloma cells. Eur. J. Biochem. 51, 283–293 (1975).

    Article  CAS  PubMed  Google Scholar 

  54. Barciszewska, M., Dirheimer, G. & Keith, G. The nucleotide sequence of methionine elongator tRNA from wheat germ. Biochem. Biophys. Res. Commun. 114, 1161–1168 (1983).

    Article  CAS  PubMed  Google Scholar 

  55. Brachmann, C. B. et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast 14, 115–132 (1998).

    Article  CAS  PubMed  Google Scholar 

  56. Alexandrov, A. et al. Rapid tRNA decay can result from lack of nonessential modifications. Mol. Cell 21, 87–96 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. De Zoysa, M. D., Wu, G., Katz, R. & Yu, Y. T. Guide-substrate base-pairing requirement for box H/ACA RNA-guided RNA pseudouridylation. RNA 24, 1106–1117 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  58. Wu, G. et al. Pseudouridines in U2 snRNA stimulate the ATPase activity of Prp5 during spliceosome assembly. EMBO J. 35, 654–667 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Rubin, G. M. Preparation of RNA and ribosomes from yeast. Methods Cell Biol. 12, 45–64 (1975).

    Article  CAS  PubMed  Google Scholar 

  60. Su, D. et al. Quantitative analysis of ribonucleoside modifications in tRNA by HPLC-coupled mass spectrometry. Nat. Protoc. 9, 828–841 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Kohrer, C. & Rajbhandary, U. L. The many applications of acid urea polyacrylamide gel electrophoresis to studies of tRNAs and aminoacyl-tRNA synthetases. Methods 44, 129–138 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  62. Sieg, J. P., Arteaga, S. J., Znosko, B. M. & Bevilacqua, P. C. MeltR software provides facile determination of nucleic acid thermodynamics. Biophys. Rep. 3, 100101 (2023).

    CAS  Google Scholar 

  63. McDowell, J. A. & Turner, D. H. Investigation of the structural basis for thermodynamic stabilities of tandem GU mismatches: solution structure of (rGAGGUCUC)2 by two-dimensional NMR and simulated annealing. Biochemistry 35, 14077–14089 (1996).

    Article  CAS  PubMed  Google Scholar 

  64. Borer, P. N. in CRC Handbook of Biochemistry and Molecular Biology, Vol. I (ed. Fasman, G. D.) 589–595 (CRC Press, 1975).

  65. Richards, E. G. in CRC Handbook of Biochemistry and Molecular Biology, Vol. I (ed. Fasman, G. D.) 596–603 (CRC Press, 1975).

  66. McGlincy, N. J. & Ingolia, N. T. Transcriptome-wide measurement of translation by ribosome profiling. Methods 126, 112–129 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Aeschimann, F., Xiong, J., Arnold, A., Dieterich, C. & Grosshans, H. Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling. Methods 85, 75–89 (2015).

    Article  CAS  PubMed  Google Scholar 

  68. Marcel, M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 17, 10–12 (2011).

    Article  Google Scholar 

  69. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15–21 (2013).

    Article  CAS  PubMed  Google Scholar 

  70. Nedialkova, D. D. & Leidel, S. A. Optimization of codon translation rates via tRNA modifications maintains proteome integrity. Cell 161, 1606–1618 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank E. M. Phizicky for extremely helpful discussions on RNA modification. We also thank B. L. Miller and A. L. Evans for sharing equipment for melting curve analysis. The LC–MS quantitative analysis of tRNA ribonucleoside modifications was carried out at the URMC MSRL. Ribosome profiling, RNA-seq and related bioinformatics analyses (partially) were carried out by TB-SEQ. This work was supported by grants R01GM138387 (to Y.-T.Y.) and R35GM145283 (to D.H.M.) from the National Institutes of Health, grant GFF521008 (to Y.-T.Y.) from the Gilbert Family Foundation, and grant 2022/45/B/ST4/03586 (to R.K.) and 2021/41/B/NZ1/03819 (to E.K.) from the National Science Center of Poland.

Author information

Authors and Affiliations

  1. Department of Biochemistry and Biophysics, Center for RNA Biology, University of Rochester Medical Center, Rochester, NY, USA

    Yi Pan, David H. Mathews & Yi-Tao Yu

  2. Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland

    Elzbieta Kierzek & Ryszard Kierzek

Authors
  1. Yi Pan
    View author publications

    Search author on:PubMed Google Scholar

  2. Elzbieta Kierzek
    View author publications

    Search author on:PubMed Google Scholar

  3. Ryszard Kierzek
    View author publications

    Search author on:PubMed Google Scholar

  4. David H. Mathews
    View author publications

    Search author on:PubMed Google Scholar

  5. Yi-Tao Yu
    View author publications

    Search author on:PubMed Google Scholar

Contributions

Y.P. and Y.-T.Y. conceptualized the study and wrote the paper. Y.P. performed most of the experiments. E.K., R.K. and D.H.M. contributed to the melting curve analysis and read and edited the manuscript. All authors approved the paper.

Corresponding author

Correspondence to Yi-Tao Yu.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Chemical Biology thanks U. Thomas Meier, Lorenzo Montanaro and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Robust expression of PTC-gRNA and the tRNA-gRNAs does not alter the level of PTC-containing cup1 mRNA.

a, Northern blot analysis showed the expression of PTC-gRNA and the tRNA-gRNAs. SNR81, an endogenous box H/ACA gRNA, and U6, a spliceosomal snRNA, served as controls. Lane M is the marker lane. b, RT-qPCR analysis showed that the level of PTC-containing mRNA was unchanged regardless of which designer gRNAs were expressed. Top, PTC(K30UAA)-cup1 mRNA; Middle, PTC(K30UAG)-cup1 mRNA; Bottom, PTC(K30UGA)-cup1 mRNA. Shown are three independent biological replicates for each sample and three technical repeats for each replicate.

Source data

Extended Data Fig. 2 Both the tRNA level and the level of charged (and uncharged) tRNA were unchanged regardless of which tRNA-gRNA was present in Saccharomyces cerevisiae.

a-f, Measurement of tRNA level using northern blot analysis. a, tRNALys(UUU); c, tRNALys(CUU); e, tRNAArg(UCU). tRNALeu(CAA) serves as a control. b, d, and f, Quantification of the data in a, c, and e, respectively. g-l, Assessment of the level of charged (and uncharged) tRNA. g, tRNALys(UUU); i, tRNALys(CUU); k, tRNAArg(UCU). h, j, and l, Quantification of the data in g, i, and k, respectively.

Source data

Extended Data Fig. 3 tRNA-gRNA does not alter the natural tRNA modification.

Nucleosides of tRNALys(CUU) from yeast cells transformed with either control tRNA-gRNA (ctrl, dark green circles) or 3 copies of tRNALys(CUU)-gRNA (3×TKc, light green squares) were quantitatively analyzed by liquid chromatography-mass spectrometry (LC-MS). All the nucleosides detected were normalized to cytidine (C) because there is no modified cytidine in yeast tRNALys(CUU). With the exception of Ψ, no differences in other nucleotide modification were detected between tRNALys(CUU) isolated from control cells and tRNALys(CUU) purified from cells transformed with tRNALys(CUU)-gRNA. The LC-MS results, coupled with the Ψ mapping data (Fig. 2c,f), indicated that tRNALys(CUU)-gRNA directed tRNALys(CUU) pseudouridylation at position 36. m1A: 1-methyladenosine; t6A: N6-threoninocarbonyladenosine; D: dihydrouridine; T: ribothymidine (5-methyluridine); m1G: 1-methylguanosine; m2G: N2-methylguanosine; m2,2G: N2,N2-dimethylguanosine; m7G: 7-methylguanosine.

Source data

Extended Data Fig. 4 Nonsense suppression assay verifying the high specificity of the Ψ-Ψ codon-anticodon pair in the mCherry context.

a,d,g, Yeast cells were co-transformed with the PTC-containing mCherry gene, either PTC(K14UAA) (a, Panels 3-10), PTC(K14UGA) (d, Panels 3-12), or PTC(K14UAG) (g, Panels 3-12), and two additional gRNA genes: two control (nonspecific) gRNAs (Panel 3), a control PTC-gRNA and a tRNALys(UUU)-gRNA (TKu) (Panel 4), a control PTC-gRNA and a tRNALys(CUU)-gRNA (TKc) (Panel 5; g, Panel 7, three copies of TKc), a control PTC-gRNA and a tRNAArg(UCU)-gRNA (TR) (Panel 6; d, Panel 7, three copies of TR), a control tRNA-gRNA and a PTC-specific gRNA (a, Panel 7; d and g, Panel 8), a PTC-specific gRNA and a tRNALys(UUU)-gRNA (TKu) (a, Panel 8; d and g, Panel 9), a PTC-specific gRNA and a tRNALys(CUU)-gRNA (TKc) (a, Panel 9; d and g, Panel 10; g, Panel 12, three copies of TKc), or a PTC-specific gRNA and a tRNAArg(UCU)-gRNA (TR) (a, Panel 10; d and g, Panel 11; d, Panel 12, three copies of TR). Exposure times are indicated (bottom). Panels 3-7 of d and g with longer exposure times are also shown. Panels 1 and 2 are positive controls: co-transformation of mCherry [wild-type (a and g) or the K14R missense variant (d)] with two control gRNAs (Panel 1) or with a control tRNA-gRNA and a PTC-specific gRNA: PTC(K14UAA)-gRNA (a, Panel 2), PTC(K14UGA)-gRNA (d, Panel 2), or PTC(K14UAG)-gRNA (g, Panel 2). Scale bar, 500μm. A robust improvement in nonsense suppression was observed only when a matched pair of gRNAs (PTC-gRNA and tRNA-gRNA) was expressed. b, e, and h, Quantification of the fluorescence data in a, d, and g, respectively. Lane numbers in b, e, and h correspond to the panel numbers in a, d, and g, respectively. Welch and Brown–Forsythe ANOVA with two-sided Welch’s t-test was performed and the sample sizes and exact p-values are shown in Source Data. c, f, and i, Western blot analysis of mCherry PTC-readthrough. The lane numbers in c, f, and i correspond to the panel numbers of a, d, and g, respectively. Lane M, marker; eEF1α, loading control.

Source data

Extended Data Fig. 5 PTC-containing mCherry mRNA level was unchanged regardless of which gRNAs were expressed in S. cerevisiae.

The legend is the same as for Extended Data Fig. 1b, except that PTC-mCherry mRNA (instead of PTC-cup1 mRNA) was used.

Source data

Extended Data Fig. 6 Introduction of a matching pair of gRNAs (PTC-gRNA and tRNA-gRNA) specifically induces PTC readthrough on the mCherryK14UAG reporter gene without affecting transcription, translation (including normal stop codon termination and the decoding of sense codons).

a, Comparison of P-site coverage on mCherryK14UAG reporter gene between the Control sample (with a control PTC-gRNA and a control tRNA-gRNA) and the Treated sample [with the PTC(K14UAG)-specific gRNA and three copies of tRNALys(CUU)-gRNA (3×TKc)]. Positions are counted so that the start codon (AUG) of the ORF has positions 1-3. The PTC and the normal stop codon are highlighted in light brown and indigo, respectively. b, Comparison of P-site coverage on four representative endogenous genes with a UAG stop codon between Control and Treated samples. Shown are the regions near the stop codons. The legend is the same as that in a. No significant readthrough was detected. Similar patterns (Control and Treated samples) were observed for other endogenous genes. c and d, Comparison of transcription (c, represented as RPKM) and translation (d, represented as PPKM; see Methods: Bioinformatics analysis for definition). ρ, Pearson correlation coefficient. No significant difference was observed between the Control and Treated samples. e and f, Log2-fold changes of codon occupancies in A-sites (e) and P-sites (f). AAG codon is highlighted in pink. No significant difference was observed between the Control and Treated samples.

Source data

Extended Data Fig. 7 Box H/ACA gRNA-directed tRNA pseudouridylation in human cells.

Total RNA was isolated from samples shown in Fig. 6. Pseudouridylation assay (CMC-modification followed by primer extension) was carried out exactly as in Fig. 2. a, a tRNALys(UUU)-specific primer was used. Left panel: Shorter exposure; Right panel: Longer exposure. b, a tRNALys(CUU)-specific primer was used. Two different exposures are shown, as in Panel a. c and d, Semi-quantification of pseudouridylation efficiency shown in a and b, respectively.

Source data

Extended Data Fig. 8 Nonsense suppression assay in the context of mCherry gene in HEK293T cells.

a and b, Monitoring of transfection efficiency using the EGFP gene. a, Transfection of wild-type mCherry gene was equally efficient regardless of the presence of different gRNAs. b, Transfection of PTC(K14UAA or K14UAG)-containing mCherry gene was similarly efficient regardless of the presence of different gRNAs. Scale bar, 100μm. c and d, Quantification of nonsense suppression in human cells shown in Fig. 6b,c, respectively. Fluorescent cells were recognized and counted. The fluorescence intensity of each cell was measured and shown as raincloud plots. e and f, Nonsense suppression of PTC(K14UGA)-containing mCherry. e, Base-pairing interactions between mCherry PTC(K14UGA)-gRNA and mCherryK14UGA and between tRNAArg(UCU)-gRNA and tRNAArg(UCU). f, Nonsense suppression was detected when HEK293T cells were co-transfected with the PTC(K14UGA)-containing mCherry gene and a PTC-specific gRNA (Panel 3). When co-transfected with an additional tRNAArg(UCU)-gRNA (TR), almost no further enhancement of nonsense suppression was detected (Panel 4). Panels 1 and 2 are controls where cells were not transfected with PTC-specific gRNA, and no nonsense suppression was detected. Transfection efficiency was the same for each transfection, as monitored by EGFP (Panels 5-8). Scale bar, 100μm. g, PTC(K14UAA or K14UAG) -mCherry mRNA level was unchanged regardless of which gRNAs were expressed in HEK293T cells. Figure legend is the same as for Extended Data Fig. 1b, except that PTC-mCherry mRNA (instead of PTC-cup1 mRNA) and HEK293T cells (instead of S. cerevisiae) were used.

Source data

Extended Data Fig. 9 Both the tRNA level and the level of charged (and uncharged) tRNA were unchanged regardless of which tRNA-gRNA was present in HEK293T cells.

a-d, Measurement of tRNA level using northern blot analysis. The results showed that the tRNA level was unchanged regardless of which tRNA-gRNA was present. a, tRNALys(UUU); c, tRNALys(CUU). tRNASer(GCU) serves as a control. b and d, Quantification of the data in a and c, respectively. e and f, Assessment of the level of charged (and uncharged) tRNA. The results show that the levels of charged (and uncharged) tRNALys(UUU) and of charged (and uncharged) tRNALys(CUU) remained unchanged regardless of which tRNA-gRNA was present (both tRNAs were almost fully charged). e, tRNALys(UUU); f, tRNALys(CUU).

Source data

Extended Data Fig. 10 Direct evidence for the genuine high-affinity Ψ-Ψ base-pair.

a, Melting curve analysis was carried out for the Ψ-Ψ and other base-pairs. A well-studied RNA duplex was used. The sequences of the two strands are shown, where X-Y stands for either the U-U, Ψ-U, Ψ-Ψ, U-A, or Ψ-A base-pair. b and c, Melting curves were measured in the 980 mM sodium chloride and 20 mM sodium phosphate buffer, pH = 7, where the RNA duplex concentration was 10 μM. The melting temperatures of the duplex with different X-Y pairs were determined.

Source data

Supplementary information

Supplementary Information

Supplementary Figs. 1–7 and Tables 1–6.

Reporting Summary

Supplementary Data 1

Statistical source data for Supplementary Fig. 4.

Source data

Source Data Fig. 1

Statistical source data.

Source Data Fig. 2

Statistical source data.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 5

Unprocessed western blots and statistical source data.

Source Data Fig. 6

Unprocessed western blots.

Source Data Extended Data Fig. 1

Statistical source data.

Source Data Extended Data Fig. 2

Unprocessed northern blots and statistical source data.

Source Data Extended Data Fig. 3

Statistical source data.

Source Data Extended Data Fig. 4

Unprocessed western blots and statistical source data.

Source Data Extended Data Fig. 5

Statistical source data.

Source Data Extended Data Fig. 6

Statistical source data.

Source Data Extended Data Fig. 7

Statistical source data.

Source Data Extended Data Fig. 8

Statistical source data.

Source Data Extended Data Fig. 9

Unprocessed northern blots and statistical source data.

Source Data Extended Data Fig. 10

Statistical source data.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pan, Y., Kierzek, E., Kierzek, R. et al. A Ψ–Ψ codon–anticodon pairing in nonsense suppression and translational recoding. Nat Chem Biol (2025). https://doi.org/10.1038/s41589-025-02025-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1038/s41589-025-02025-9

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing