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Converting nonsense codons into sense codons by targeted pseudouridylation

Abstract

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.

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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.

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Acknowledgements

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.

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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.

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Correspondence to Yi-Tao Yu.

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The authors declare no competing financial interests.

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Karijolich, J., Yu, YT. Converting nonsense codons into sense codons by targeted pseudouridylation. Nature 474, 395–398 (2011). https://doi.org/10.1038/nature10165

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