Skip to main content

Thank you for visiting 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.

Converting nonsense codons into sense codons by targeted pseudouridylation


All three translation termination codons, or nonsense codons, contain a uridine residue at the first position of the codon1,2,3. Here, we demonstrate that pseudouridylation (conversion of uridine into pseudouridine (Ψ), ref. 4) of nonsense codons suppresses translation termination both in vitro and in vivo. In vivo targeting of nonsense codons is accomplished by the expression of an H/ACA RNA capable of directing the isomerization of uridine to Ψ within the nonsense codon. Thus, targeted pseudouridylation represents a novel approach for promoting nonsense suppression in vivo. Remarkably, we also show that pseudouridylated nonsense codons code for amino acids with similar properties. Specifically, ΨAA and ΨAG code for serine and threonine, whereas ΨGA codes for tyrosine and phenylalanine, thus suggesting a new mode of decoding. Our results also suggest that RNA modification, as a naturally occurring mechanism, may offer a new way to expand the genetic code.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Pseudouridylation of a termination codon promotes nonsense suppression in vitro.
Figure 2: Quantification of cup1-PTC pseudouridylation.
Figure 3: Expression of an H/ACA RNA targeting the PTC of cup-PTC for pseudouridylation promotes nonsense suppression.
Figure 4: Generalization of Ψ-mediated nonsense suppression and determination of amino acids coded for by pseudouridylated nonsense codons.


  1. 1

    Brenner, S., Barnett, L., Katz, E. R. & Crick, F. H. UGA: a third nonsense triplet in the genetic code. Nature 213, 449–450 (1967)

    ADS  CAS  Article  Google Scholar 

  2. 2

    Brenner, S., Stretton, A. O. & Kaplan, S. Genetic code: the ‘nonsense’ triplets for chain termination and their suppression. Nature 206, 994–998 (1965)

    ADS  CAS  Article  Google Scholar 

  3. 3

    Weigert, M. G. & Garen, A. Base composition of nonsense codons in E. coli. Evidence from amino-acid substitutions at a tryptophan site in alkaline phosphatase. Nature 206, 992–994 (1965)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Cohn, W. E. 5-Ribosyl uracil, a carbon-carbon ribofuranosyl nucleoside in ribonucleic acids. Biochim. Biophys. Acta 32, 569–571 (1959)

    CAS  Article  Google Scholar 

  5. 5

    Charette, M. & Gray, M. W. Pseudouridine in RNA: what, where, how, and why. IUBMB Life 49, 341051 (2000)

    Google Scholar 

  6. 6

    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)

    CAS  PubMed  PubMed Central  Google Scholar 

  7. 7

    Hamer, D. H., Thiele, D. J. & Lemontt, J. E. Function and autoregulation of yeast copperthionein. Science 228, 685–690 (1985)

    ADS  CAS  Article  Google Scholar 

  8. 8

    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)

    CAS  Article  Google Scholar 

  9. 9

    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)

    CAS  Article  Google Scholar 

  10. 10

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

    CAS  Article  Google Scholar 

  11. 11

    Leeds, P., Peltz, S. W., Jacobson, A. & Culbertson, M. R. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 5, 2303–2314 (1991)

    CAS  Article  Google Scholar 

  12. 12

    Leeds, P., Wood, J. M., Lee, B. S. & Culbertson, M. R. Gene products that promote mRNA turnover in Saccharomyces cerevisiae. Mol. Cell. Biol. 12, 2165–2177 (1992)

    CAS  Article  Google Scholar 

  13. 13

    Wilusz, C. J., Wang, W. & Peltz, S. W. Curbing the nonsense: the activation and regulation of mRNA surveillance. Genes Dev. 15, 1781–1785 (2001)

    Google Scholar 

  14. 14

    Agris, P. F. The importance of being modified: roles of modified nucleosides and Mg2+ in RNA structure and function. Prog. Nucleic Acid Res. Mol. Biol. 53, 79–129 (1996)

    CAS  Article  Google Scholar 

  15. 15

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

    CAS  Article  Google Scholar 

  16. 16

    Auffinger, P. & Westhof, E. RNA hydration: three nanoseconds of multiple molecular dynamics simulations of the solvated tRNAAsp anticodon hairpin. J. Mol. Biol. 269, 326–341 (1997)

    CAS  Article  Google Scholar 

  17. 17

    Agris, P. F. Decoding the genome: a modified view. Nucleic Acids Res. 32, 223–238 (2004)

    CAS  Article  Google Scholar 

  18. 18

    Song, H. et al. The crystal structure of human eukaryotic release factor eRF1—mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100, 311–321 (2000)

    CAS  Article  Google Scholar 

  19. 19

    Atkins, J. F., Gesteland, R. F., Reid, B. R. & Anderson, C. W. Normal tRNAs promote ribosomal frameshifting. Cell 18, 1119–1131 (1979)

    CAS  Article  Google Scholar 

  20. 20

    Frischmeyer, P. A. & Dietz, H. C. Nonsense-mediated mRNA decay in health and disease. Hum. Mol. Genet. 8, 1893–1900 (1999)

    CAS  Article  Google Scholar 

  21. 21

    Wu, G., Xiao, M., Yang, C. & Yu, Y. T. U2 snRNA is inducibly pseudouridylated at novel sites by Pus7p and snR81 RNP. EMBO J. 30, 79–89 (2010)

    CAS  Article  Google Scholar 

  22. 22

    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)

    CAS  Article  Google Scholar 

  23. 23

    Chernyakov, I., Whipple, J. M., Kotelawala, L., Grayhack, E. J. & Phizicky, E. M. Degradation of several hypomodified mature tRNA species in Saccharomyces cerevisiae is mediated by Met22 and the 5′-3′ exonucleases Rat1 and Xrn1. Genes Dev. 22, 1369–1380 (2008)

    CAS  Article  Google Scholar 

  24. 24

    Yu, Y. T. Site-specific 4-thiouridine incorporation into RNA molecules. Methods Enzymol. 318, 71–88 (2000)

    CAS  Article  Google Scholar 

  25. 25

    Zhao, X. & Yu, Y. T. Pseudouridines in and near the branch site recognition region of U2 snRNA are required for snRNP biogenesis and pre-mRNA splicing in Xenopus oocytes. RNA 10, 681–690 (2004)

    CAS  Article  Google Scholar 

  26. 26

    Gelperin, D. M. et al. Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev. 19, 2816–2826 (2005)

    CAS  Article  Google Scholar 

  27. 27

    Zebarjadian, Y., King, T., Fournier, M. J., Clarke, L. & Carbon, J. Point mutations in yeast CBF5 can abolish in vivo pseudouridylation of rRNA. Mol. Cell. Biol. 19, 7461–7472 (1999)

    CAS  Article  Google Scholar 

Download references


We thank F. Hagen and the Proteomics Core at the University of Rochester for performing the mass spectrometry analysis. We also thank E. Phizicky and B. Grayhack for the wild-type TRM4 construct, C. Guthrie for the cup1Δ yeast strain, D. Mcpheeters for the wild-type CUP1 construct, and M. Dumont for the anti-Eno1p antibody. Lastly, we would like to thank members of the Yu laboratory, especially X. Zhao, for helpful discussions.

Author information




J.K. and Y.-T.Y. designed and interpreted the experiments. Mass spectrometry was performed at the Proteomics Core at the University of Rochester Medical Center. J.K. performed all other experiments.

Corresponding author

Correspondence to Yi-Tao Yu.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-10 with legends. (PDF 1570 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


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