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Intracellular calcium regulates nonsense-mediated mRNA decay.

Nickless A, Jackson E, Marasa J, Nugent P, Mercer RW, Piwnica-Worms D, You Z - Nat. Med. (2014)

Bottom Line: Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells.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.Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.

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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A high-throughput screen using the NMD reporter identified existing drugs that modulate NMD(a) Procedure for a high-throughput screen of the Pharmakon 1600 drug library.(b) Primary screening data. Data are shown as the log2 of the normalized CBR/CBG ratio for each compound. Each ratio was normalized to the DMSO controls on the same plate. Data represent the average of three biological replicates. Compounds were ordered from left to right with increasing relative CBR/CBG ratios. Eight candidate NMD inhibitors of stringent statistical significance are shown in the table. See Supplementary Table 2 for primary screen data of all compounds in the library.
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Figure 2: A high-throughput screen using the NMD reporter identified existing drugs that modulate NMD(a) Procedure for a high-throughput screen of the Pharmakon 1600 drug library.(b) Primary screening data. Data are shown as the log2 of the normalized CBR/CBG ratio for each compound. Each ratio was normalized to the DMSO controls on the same plate. Data represent the average of three biological replicates. Compounds were ordered from left to right with increasing relative CBR/CBG ratios. Eight candidate NMD inhibitors of stringent statistical significance are shown in the table. See Supplementary Table 2 for primary screen data of all compounds in the library.

Mentions: Using the reporter system described above, we next performed a high-throughput screen to identify drug candidates that can alter NMD activity in human cells. The Pharmakon library, which contains a diverse array of 1,600 clinically-evaluated compounds, was used as the source of small molecules in the hope of repurposing existing drug candidates to fast-track the drug development process. Human U2OS cells containing the dual-colored NMD reporter were seeded on 96-well plates and individual drugs were added to each well at a concentration of 10 µM, along with appropriate controls. Twenty-four hours after drug treatment, D-luciferin was added to the medium followed by bioluminescence imaging and spectral unmixing to obtain CBR and CBG signals (Fig. 2a). A Z’ factor of 0.77 was calculated for the reporter assay, demonstrating the robustness of the strategy (see Methods). As shown in Fig. 2b and Supplementary Table 2, the majority of the compounds in the library exhibited little or no effect on NMD of the reporter. However, 8 candidate inhibitors and 14 candidate enhancers of NMD were identified by quartile analysis after employing stringent criteria for hit selection (Fig. 2b and Supplementary Table 3)13. Focusing on NMD inhibitors in this study, the effects of seven of the inhibitor hits were confirmed in follow-up analysis, and each validated compound inhibited NMD in a dose-dependent manner (data not shown). Strikingly, the top five verified hits, including digitoxin, digoxin, lanatoside C, proscillaridin and ouabain, are all cardiac glycosides (CGs), which inhibit Na+/K+-ATPase in cells (Fig. 2b)14.


Intracellular calcium regulates nonsense-mediated mRNA decay.

Nickless A, Jackson E, Marasa J, Nugent P, Mercer RW, Piwnica-Worms D, You Z - Nat. Med. (2014)

A high-throughput screen using the NMD reporter identified existing drugs that modulate NMD(a) Procedure for a high-throughput screen of the Pharmakon 1600 drug library.(b) Primary screening data. Data are shown as the log2 of the normalized CBR/CBG ratio for each compound. Each ratio was normalized to the DMSO controls on the same plate. Data represent the average of three biological replicates. Compounds were ordered from left to right with increasing relative CBR/CBG ratios. Eight candidate NMD inhibitors of stringent statistical significance are shown in the table. See Supplementary Table 2 for primary screen data of all compounds in the library.
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Related In: Results  -  Collection

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Figure 2: A high-throughput screen using the NMD reporter identified existing drugs that modulate NMD(a) Procedure for a high-throughput screen of the Pharmakon 1600 drug library.(b) Primary screening data. Data are shown as the log2 of the normalized CBR/CBG ratio for each compound. Each ratio was normalized to the DMSO controls on the same plate. Data represent the average of three biological replicates. Compounds were ordered from left to right with increasing relative CBR/CBG ratios. Eight candidate NMD inhibitors of stringent statistical significance are shown in the table. See Supplementary Table 2 for primary screen data of all compounds in the library.
Mentions: Using the reporter system described above, we next performed a high-throughput screen to identify drug candidates that can alter NMD activity in human cells. The Pharmakon library, which contains a diverse array of 1,600 clinically-evaluated compounds, was used as the source of small molecules in the hope of repurposing existing drug candidates to fast-track the drug development process. Human U2OS cells containing the dual-colored NMD reporter were seeded on 96-well plates and individual drugs were added to each well at a concentration of 10 µM, along with appropriate controls. Twenty-four hours after drug treatment, D-luciferin was added to the medium followed by bioluminescence imaging and spectral unmixing to obtain CBR and CBG signals (Fig. 2a). A Z’ factor of 0.77 was calculated for the reporter assay, demonstrating the robustness of the strategy (see Methods). As shown in Fig. 2b and Supplementary Table 2, the majority of the compounds in the library exhibited little or no effect on NMD of the reporter. However, 8 candidate inhibitors and 14 candidate enhancers of NMD were identified by quartile analysis after employing stringent criteria for hit selection (Fig. 2b and Supplementary Table 3)13. Focusing on NMD inhibitors in this study, the effects of seven of the inhibitor hits were confirmed in follow-up analysis, and each validated compound inhibited NMD in a dose-dependent manner (data not shown). Strikingly, the top five verified hits, including digitoxin, digoxin, lanatoside C, proscillaridin and ouabain, are all cardiac glycosides (CGs), which inhibit Na+/K+-ATPase in cells (Fig. 2b)14.

Bottom Line: Here, we have developed a new multicolored bioluminescence-based reporter system that can specifically and effectively assay NMD in live human cells.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.Thus, this study reveals intracellular calcium as a key regulator of NMD and has implications for exploiting NMD in the treatment of disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology &Physiology, Washington University School of Medicine, St. Louis, Missouri, USA.

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.

Show MeSH
Related in: MedlinePlus