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Intracellular calcium regulates nonsense-mediated mRNA decay

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Abstract

The nonsense-mediated mRNA decay (NMD) pathway selectively eliminates aberrant transcripts containing premature translation termination codons and regulates the levels of a number of physiological mRNAs. NMD modulates the clinical outcome of a variety of human diseases, including cancer and many genetic disorders, and may represent a target for therapeutic intervention. Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells. Using this reporter system, we conducted a robust high-throughput small-molecule screen in human cells and, unpredictably, identified a group of cardiac glycosides, including ouabain and digoxin, as potent inhibitors of NMD. Cardiac glycoside–mediated effects on NMD are dependent on binding and inhibiting the sodium-potassium ATPase on the plasma membrane and subsequent elevation of intracellular calcium levels. Induction of calcium release from the endoplasmic reticulum also leads to inhibition of NMD. Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

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Figure 1: A dual-color bioluminescence-based NMD reporter system.
Figure 2: A high-throughput screen using the NMD reporter identified existing drugs that modulate NMD.
Figure 3: CGs are potent inhibitors of NMD.
Figure 4: CGs block NMD through inhibition of Na+/K+-ATPase and elevation of intracellular calcium.

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Acknowledgements

We thank P. Wu and X. Chen for their contributions in the early stage of this project. We are grateful to O. Mühlemann (University of Bern) for providing the pβ510 reporter construct, which we used to obtain the TRB minigene reporter sequence for our reporter construction, and R. Mecham (Washington University School of Medicine) for providing primary mouse skin fibroblasts. This study was supported by a Molecular Imaging Center grant from the US National Institutes of Health to Washington University and The University of Texas MD Anderson Cancer Center (P50 CA94056, D.P.-W.), by a Washington University Molecular Imaging Center Pilot Research Project grant (Z.Y.), by a grant from the US National Institutes of Health (R01GM098535, Z.Y.) and by an Interdisciplinary Research Initiative grant from the Children's Discovery Institute of Washington University (MC-II-2012-215, Z.Y. and D.P.-W.). The High-Throughput Screening Core at the Washington University School of Medicine is supported in part by the Siteman Cancer Center (Cancer Center Support grant P30 CA91842 from the US National Cancer Institute).

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Contributions

Z.Y. and D.P.-W. conceived the project and supervised the studies. A.N. and Z.Y. designed and performed the experiments. E.J. contributed to the bioluminescence imaging and the analysis of screen and imaging data. J.M. assisted with the high-throughput chemical screen. P.N. assisted with the analysis of the screen results. R.W.M. provided expression constructs for rat Na+/K+-ATPase α subunits and technical advice. A.N., Z.Y. and D.P.-W. wrote the manuscript.

Corresponding authors

Correspondence to David Piwnica-Worms or Zhongsheng You.

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The authors declare no competing financial interests.

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Nickless, A., Jackson, E., Marasa, J. et al. Intracellular calcium regulates nonsense-mediated mRNA decay. Nat Med 20, 961–966 (2014). https://doi.org/10.1038/nm.3620

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