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
Open Access articles citing this article.
Nature Communications Open Access 27 May 2022
Molecular Cancer Open Access 29 April 2021
Cell & Bioscience Open Access 19 May 2017
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Goldmann, T. et al. EMBO Mol. Med. 4, 1186–1199 (2012).
Rigo, F., Hua, Y., Krainer, A.R. & Bennett, C.F. J. Cell Biol. 199, 21–25 (2012).
Popp, M.W.-L. & Maquat, L.E. Annu. Rev. Genet. 47, 139–165 (2013).
Keeling, K.M. et al. PLoS One 8, e60478 (2013).
Keeling, K.M. & Bedwell, D.M. Wiley Interdiscip. Rev. RNA 2, 837–852 (2011).
Bhuvanagiri, M. et al. EMBO Mol. Med. 6, 1593–1609 (2014).
Martin, L. et al. Cancer Res. 74, 3104–3113 (2014).
Ni, J.Z. et al. Genes Dev. 21, 708–718 (2007).
Karam, R. & Wilkinson, M. RNA Biol. 9, 22–26 (2012).
Jolly, L.A., Homan, C.C., Jacob, R., Barry, S. & Gecz, J. Hum. Mol. Genet. 22, 4673–4687 (2013).
Shibuya, T., Tange, T.O., Sonenberg, N. & Moore, M.J. Nat. Struct. Mol. Biol. 11, 346–351 (2004).
Kole, R., Krainer, A.R. & Altman, S. Nat. Rev. Drug Discov. 11, 125–140 (2012).
Giorgi, C. & Moore, M.J. Semin. Cell Dev. Biol. 18, 186–193 (2007).
Trecartin, R.F. et al. J. Clin. Invest. 68, 1012–1017 (1981).
Pan, Q. et al. Genes Dev. 20, 153–158 (2006).
Gardner, L.B. Mol. Cancer Res. 8, 295–308 (2010).
Winkler, J., Stessl, M., Amartey, J. & Noe, C.R. ChemMedChem 5, 1344–1352 (2010).
Zhang, Z. & Krainer, A.R. Proc. Natl. Acad. Sci. USA 104, 11574–11579 (2007).
Trcek, T., Sato, H., Singer, R.H. & Maquat, L.E. Genes Dev. 27, 541–551 (2013).
Keeling, K.M., Xue, X., Gunn, G. & Bedwell, D.M. Annu. Rev. Genomics Hum. Genet. 15, 371–394 (2014).
Loughran, G. et al. Nucleic Acids Res. 42, 8928–8938 (2014).
Baker, B.F. et al. J. Biol. Chem. 272, 11994–12000 (1997).
Jeong, J.-Y. et al. Appl. Environ. Microbiol. 78, 5440–5443 (2012).
Hossain, M. & Stillman, B. Genes Dev. 26, 1797–1810 (2012).
Mayeda, A. & Krainer, A.R. Methods Mol. Biol. 118, 315–321 (1999).
Mayeda, A. & Krainer, A.R. Methods Mol. Biol. 118, 309–314 (1999).
Zheng, W., Finkel, J.S., Landers, S.M., Long, R.M. & Culbertson, M.R. Genetics 180, 1391–1405 (2008).
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.
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 3 HBB (WT) expression is not altered by treatment with H-24 ASO (50 nM; n = 3 independent transfections).
MECP2 (S65X) undergoes efficient NMD, resulting in a 95% reduction in mRNA level, compared to the wild-type mRNA (n = 3 independent samples).
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.
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.
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).
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.
About this article
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
This article is cited by
Nature Communications (2022)
Molecular Cancer (2021)
Nature Reviews Drug Discovery (2021)
Nature Reviews Drug Discovery (2020)
Nature Reviews Genetics (2018)