Skip to main content

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.

  • Brief Communication
  • Published:

Antisense oligonucleotide–directed inhibition of nonsense-mediated mRNA decay

Nature Biotechnology volume 34, pages 164–166 (2016)Cite this article

Subjects

Abstract

Nonsense-mediated mRNA decay (NMD) is a cellular quality-control mechanism that is thought to exacerbate the phenotype of certain pathogenic nonsense mutations by preventing the expression of semi-functional proteins. NMD also limits the efficacy of read-through compound (RTC)-based therapies. Here, we report a gene-specific method of NMD inhibition using antisense oligonucleotides (ASOs) and combine this approach with an RTC to effectively restore the expression of full-length protein from a nonsense-mutant allele.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Design and testing of EJC-targeting ASOs.
Figure 2: Validation of the mechanism of action of NMD inhibition.

Similar content being viewed by others

References

  1. Goldmann, T. et al. EMBO Mol. Med. 4, 1186–1199 (2012).

    Article  CAS  Google Scholar 

  2. Rigo, F., Hua, Y., Krainer, A.R. & Bennett, C.F. J. Cell Biol. 199, 21–25 (2012).

    Article  CAS  Google Scholar 

  3. Popp, M.W.-L. & Maquat, L.E. Annu. Rev. Genet. 47, 139–165 (2013).

    Article  CAS  Google Scholar 

  4. Keeling, K.M. et al. PLoS One 8, e60478 (2013).

    Article  CAS  Google Scholar 

  5. Keeling, K.M. & Bedwell, D.M. Wiley Interdiscip. Rev. RNA 2, 837–852 (2011).

    Article  CAS  Google Scholar 

  6. Bhuvanagiri, M. et al. EMBO Mol. Med. 6, 1593–1609 (2014).

    Article  CAS  Google Scholar 

  7. Martin, L. et al. Cancer Res. 74, 3104–3113 (2014).

    Article  CAS  Google Scholar 

  8. Ni, J.Z. et al. Genes Dev. 21, 708–718 (2007).

    Article  CAS  Google Scholar 

  9. Karam, R. & Wilkinson, M. RNA Biol. 9, 22–26 (2012).

    Article  CAS  Google Scholar 

  10. Jolly, L.A., Homan, C.C., Jacob, R., Barry, S. & Gecz, J. Hum. Mol. Genet. 22, 4673–4687 (2013).

    Article  CAS  Google Scholar 

  11. Shibuya, T., Tange, T.O., Sonenberg, N. & Moore, M.J. Nat. Struct. Mol. Biol. 11, 346–351 (2004).

    Article  CAS  Google Scholar 

  12. Kole, R., Krainer, A.R. & Altman, S. Nat. Rev. Drug Discov. 11, 125–140 (2012).

    Article  CAS  Google Scholar 

  13. Giorgi, C. & Moore, M.J. Semin. Cell Dev. Biol. 18, 186–193 (2007).

    Article  CAS  Google Scholar 

  14. Trecartin, R.F. et al. J. Clin. Invest. 68, 1012–1017 (1981).

    Article  CAS  Google Scholar 

  15. Pan, Q. et al. Genes Dev. 20, 153–158 (2006).

    Article  CAS  Google Scholar 

  16. Gardner, L.B. Mol. Cancer Res. 8, 295–308 (2010).

    Article  CAS  Google Scholar 

  17. Winkler, J., Stessl, M., Amartey, J. & Noe, C.R. ChemMedChem 5, 1344–1352 (2010).

    Article  CAS  Google Scholar 

  18. Zhang, Z. & Krainer, A.R. Proc. Natl. Acad. Sci. USA 104, 11574–11579 (2007).

    Article  CAS  Google Scholar 

  19. Trcek, T., Sato, H., Singer, R.H. & Maquat, L.E. Genes Dev. 27, 541–551 (2013).

    Article  CAS  Google Scholar 

  20. Keeling, K.M., Xue, X., Gunn, G. & Bedwell, D.M. Annu. Rev. Genomics Hum. Genet. 15, 371–394 (2014).

    Article  CAS  Google Scholar 

  21. Loughran, G. et al. Nucleic Acids Res. 42, 8928–8938 (2014).

    Article  CAS  Google Scholar 

  22. Baker, B.F. et al. J. Biol. Chem. 272, 11994–12000 (1997).

    Article  CAS  Google Scholar 

  23. Jeong, J.-Y. et al. Appl. Environ. Microbiol. 78, 5440–5443 (2012).

    Article  CAS  Google Scholar 

  24. Hossain, M. & Stillman, B. Genes Dev. 26, 1797–1810 (2012).

    Article  CAS  Google Scholar 

  25. Mayeda, A. & Krainer, A.R. Methods Mol. Biol. 118, 315–321 (1999).

    CAS  PubMed  Google Scholar 

  26. Mayeda, A. & Krainer, A.R. Methods Mol. Biol. 118, 309–314 (1999).

    CAS  PubMed  Google Scholar 

  27. Zheng, W., Finkel, J.S., Landers, S.M., Long, R.M. & Culbertson, M.R. Genetics 180, 1391–1405 (2008).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank F. Bennett for helpful discussions. This work was supported by US National Institutes of Health (NIH) grants R21-NS081448 and R37-GM042699 and by a research grant from the University of Pennsylvania's Center for Orphan Disease Research and Therapy to A.R.K. T.T.N. was supported by NIH grants T32GM008444 and F31NS087747. We acknowledge assistance from Cold Spring Harbor Laboratory Shared Resources, funded in part by Cancer Center Support Grant 5P30CA045508.

Author information

Authors and Affiliations

  1. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, USA

    Tomoki T Nomakuchi, Isabel Aznarez & Adrian R Krainer

  2. Stony Brook University School of Medicine, Stony Brook, New York, USA

    Tomoki T Nomakuchi

  3. Isis Pharmaceuticals, Carslbad, California, USA

    Frank Rigo

Authors
  1. Tomoki T Nomakuchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Frank Rigo
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Isabel Aznarez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Adrian R Krainer
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

T.T.N., I.A. and A.R.K. designed the experiments. I.A. and F.R. provided critical reagents. T.T.N. performed the research. T.T.N. and A.R.K. wrote the manuscript. All authors approved the manuscript.

Corresponding authors

Correspondence to Isabel Aznarez or Adrian R Krainer.

Ethics declarations

Competing interests

T.T.N., I.A. and A.R.K. have filed a patent application (PCT/US2014/054151) on this technology; F.R. is an employee of and A.R.K. is a consultant and collaborator for Isis Pharmaceuticals.

Integrated supplementary information

Supplementary Figure 1 Baseline expression levels comparing wild-type (WT) and three variants of the T39-PTC HBB constructs.

T39 and T39+24 mRNAs were reduced by approximately 95%, and T39(UGAC) was reduced by approximately 85%. n = 3 independent transfections, error bars represent s.d.

Supplementary Figure 2 Uncropped autoradiograph corresponding to Figure 1c.

Supplementary Figure 3 HBB (WT) expression is not altered by treatment with H-24 ASO (50 nM; n = 3 independent transfections).

Supplementary Figure 4 Baseline expression levels of MECP2 wild type (WT) and S65X mRNA.

MECP2 (S65X) undergoes efficient NMD, resulting in a 95% reduction in mRNA level, compared to the wild-type mRNA (n = 3 independent samples).

Supplementary Figure 5 Screen of 19 ASOs targeting the last EJC of MECP2.

100 nM of each ASO was transfected, expression of the stably integrated minigene was induced with 1 µg/ml tetracycline at 6 hr post-transfection, and RNA was isolated at 48 hr post-transfection. mRNA was measured by radioactive RT-PCR and quantitated on a phosphorimager. The fold change relative to no-ASO transfection is indicated below each lane.

Supplementary Figure 6 NMD inhibition by ASOs is specific to the targeted transcript.

Four known endogenous NMD targets15 were tested and remained essentially unchanged upon H-24 ASO treatment of U2OS cells expressing HBB (T39) or M-33 ASO treatment of U2OS cells expressing MECP2 (S65X) cells. The difference in expression levels of the transcripts before and after a 5-hr treatment with the translation inhibitor cycloheximide (CHX; 100 µg/ml) was tested in MECP2 (S65X) expressing cells (top left) to confirm their susceptibility to NMD, which requires translation. n = 3 independent samples, *P < 0.05.

Supplementary Figure 7 Representative autoradiographs corresponding to Figure 1g.

Supplementary Figure 8 Transcript subject to NMD is stabilized by ASO treatment.

HBB-T39 mRNA decay rate after actinomycin D treatment (5 µg/ml) decreased when cells were pretreated with 50 nM H-24 ASO. *P <0.05; n = 3 independent transfections.

Supplementary Figure 9 EJC-targeting ASOs increase the amount of protein expressed from mRNAs targeted by NMD.

U2OS cells expressing N-terminal GFP-tagged HBB (T39) were transfected with 100 nM H-24 ASO. Western blotting with anti-GFP antibody confirms the increase in truncated protein expression upon NMD inhibition (P < 0.01; n = 3 independent transfections). Numbers below the panel represent the fold changes ± s.d., as calculated by densitometry.

Supplementary Figure 10 N-terminal GFP-labeled wild-type HBB protein expression is not significantly altered by ASO treatment (50 nM, n = 3 independent transfections).

Supplementary Figure 11 Representative western blot corresponding to Figure 2d.

The T39 stop codon (TAG) plus 4 nt downstream were replaced with TGACTAG to promote a low level of spontaneous translational read-through. Anti-GFP antibody detects the truncated GFP-HBB, as well as the full-length read-through product. The cells were transfected with 50 nM of the indicated ASOs, and incubated with or without 1 mg/ml G418.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–11, Supplementary Tables 1 and 2 (PDF 1211 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nomakuchi, T., Rigo, F., Aznarez, I. et al. Antisense oligonucleotide–directed inhibition of nonsense-mediated mRNA decay. Nat Biotechnol 34, 164–166 (2016). https://doi.org/10.1038/nbt.3427

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt.3427

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