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Inducing nonsense suppression by targeted pseudouridylation

Nature Protocols volume 7, pages 789–800 (2012)Cite this article

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Abstract

Isomerization from uridine to pseudouridine (pseudouridylation) is largely catalyzed by a family of small ribonucleoproteins called box H/ACA RNPs, each of which contains one unique small RNA—the box H/ACA RNA. The specificity of the pseudouridylation reaction is determined by the base-pairing interactions between the guide sequence of the box H/ACA RNA and the target sequence within an RNA substrate. Thus, by creating a new box H/ACA RNA harboring an artificial guide sequence that base-pairs with the substrate sequence, one can site-specifically introduce pseudouridines into virtually any RNA (e.g., mRNA, ribosomal RNA, small nuclear RNA, telomerase RNA and so on). Pseudouridylation changes the properties of a uridine residue and is likely to alter the role of its corresponding RNA in certain cellular processes, thereby enabling basic research into the effects of RNA modifications. Here we take a TRM4 reporter gene (also known as NCL1) as an example, and we present a protocol for designing a box H/ACA RNA to site-specifically pseudouridylate TRM4 mRNA. Disease-related mutation can result in early termination of translation by creating a premature termination codon (PTC); however, pseudouridylation at the PTC can suppress this translation termination (nonsense suppression). Thus, the experimental procedures described in this protocol may provide a novel way to treat PTC-related diseases. This protocol takes 10–13 d to complete.

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Figure 1: Schematic representation of Ψ formation and an artificial box H/ACA RNA.
Figure 2: Schematic representation of the construction of the PTC-containing TRM4 expression cassette.
Figure 3: Amino acid sequence of Trm4p.
Figure 4: PCR strategy for generating artificial snR81.
Figure 5: Western blot analysis of TRM4-PTC nonsense suppression.

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Acknowledgements

We thank the members of the Yu laboratory for discussion and inspiration. We also thank E. Phizicky, E. Grayhack and M. Dumont (University of Rochester Medical Center) for providing some of the reagents used in this work, and F. Hagen for helping analyze mass spectrometry data.

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, New York, USA

    Chao Huang, Guowei Wu & Yi-Tao Yu

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  1. Chao Huang
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Contributions

C.H., G.W. and Y.-T.Y. prepared the figures and wrote the manuscript.

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Correspondence to Yi-Tao Yu.

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Huang, C., Wu, G. & Yu, YT. Inducing nonsense suppression by targeted pseudouridylation. Nat Protoc 7, 789–800 (2012). https://doi.org/10.1038/nprot.2012.029

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