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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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Cardiac glycosides are potent inhibitors of NMD(a) CBG bioluminescence signal (lower panel) and ratios of CBR/CBG bioluminescence signals (upper panel) in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the indicated concentrations. The CBG signal and the CBR/CBG ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 (paired t-test).(b) Western blot analysis of CBR-TCR(PTC) and CBG-TCR(WT) protein levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a.(c) Ratios of CBR-TCR(PTC)/CBG-TCR(WT) mRNA levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of four independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(d) Effects of cardiac glycosides on the stability of the PTC-containing p53 mRNA in Calu-6 cells. Cells were treated with DMSO or cardiac glycosides at the concentrations indicated in a for 16 h before the addition of the transcriptional inhibitor actinomycin D (5 µg ml−1) to block new RNA synthesis. Total RNA was collected immediately before or 6 h after the addition of actinomycin D, and p53 mRNA levels were analyzed by RT-qPCR and normalized to GAPDH mRNA levels. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001(paired t-test).(e) Effects of ouabain on the stability of wild-type endogenous NMD target transcripts (UPP1, ATF-4, Pim3 and Pisd) in Calu-6 cells. ORCL mRNA, which is not a NMD target, was used as a control. Cells were treated with DMSO or ouabain, and then actinomycin D; samples were collected and analyzed as described in d. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from four independent experiments. **P < 0.01 (paired t-test).
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Figure 3: Cardiac glycosides are potent inhibitors of NMD(a) CBG bioluminescence signal (lower panel) and ratios of CBR/CBG bioluminescence signals (upper panel) in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the indicated concentrations. The CBG signal and the CBR/CBG ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 (paired t-test).(b) Western blot analysis of CBR-TCR(PTC) and CBG-TCR(WT) protein levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a.(c) Ratios of CBR-TCR(PTC)/CBG-TCR(WT) mRNA levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of four independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(d) Effects of cardiac glycosides on the stability of the PTC-containing p53 mRNA in Calu-6 cells. Cells were treated with DMSO or cardiac glycosides at the concentrations indicated in a for 16 h before the addition of the transcriptional inhibitor actinomycin D (5 µg ml−1) to block new RNA synthesis. Total RNA was collected immediately before or 6 h after the addition of actinomycin D, and p53 mRNA levels were analyzed by RT-qPCR and normalized to GAPDH mRNA levels. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001(paired t-test).(e) Effects of ouabain on the stability of wild-type endogenous NMD target transcripts (UPP1, ATF-4, Pim3 and Pisd) in Calu-6 cells. ORCL mRNA, which is not a NMD target, was used as a control. Cells were treated with DMSO or ouabain, and then actinomycin D; samples were collected and analyzed as described in d. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from four independent experiments. **P < 0.01 (paired t-test).

Mentions: At the initial screening concentration (10 µM), all CGs increased CBR signal compared to DMSO treatment, but these drugs also decreased CBG signal, potentially due to non-specific toxic effects on cell viability (Supplementary Table 2). However, we identified a lower concentration for each drug at which the CBR activity was still dramatically increased while the CBG activity remained largely unaffected (Fig. 3a). At these modest concentrations, these drugs did not significantly impact general translation in cells and had only a mild inhibitory effect on cell viability (Supplementary Fig. 3a, b). Because CBR and CBG proteins are relatively stable, the inhibitory effects of CGs on the signal generated by the NMD reporter was time-dependent, increasing over the 24 h treatment period; CG-mediated blockade of NMD was fully concentration-dependent, showing reporter-generated EC50 values of ~70 nM and ~200 nM for ouabain and digoxin, respectively (Supplementary Fig. 4a, b). Importantly, our bioluminescence imaging results were corroborated by Western blot and reporter-specific RT-qPCR analyses of the CBR-TCR(PTC) and CBG-TCR(WT) protein and mRNA levels, respectively (Fig. 3b, c).


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)

Cardiac glycosides are potent inhibitors of NMD(a) CBG bioluminescence signal (lower panel) and ratios of CBR/CBG bioluminescence signals (upper panel) in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the indicated concentrations. The CBG signal and the CBR/CBG ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 (paired t-test).(b) Western blot analysis of CBR-TCR(PTC) and CBG-TCR(WT) protein levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a.(c) Ratios of CBR-TCR(PTC)/CBG-TCR(WT) mRNA levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of four independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(d) Effects of cardiac glycosides on the stability of the PTC-containing p53 mRNA in Calu-6 cells. Cells were treated with DMSO or cardiac glycosides at the concentrations indicated in a for 16 h before the addition of the transcriptional inhibitor actinomycin D (5 µg ml−1) to block new RNA synthesis. Total RNA was collected immediately before or 6 h after the addition of actinomycin D, and p53 mRNA levels were analyzed by RT-qPCR and normalized to GAPDH mRNA levels. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001(paired t-test).(e) Effects of ouabain on the stability of wild-type endogenous NMD target transcripts (UPP1, ATF-4, Pim3 and Pisd) in Calu-6 cells. ORCL mRNA, which is not a NMD target, was used as a control. Cells were treated with DMSO or ouabain, and then actinomycin D; samples were collected and analyzed as described in d. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from four independent experiments. **P < 0.01 (paired t-test).
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Figure 3: Cardiac glycosides are potent inhibitors of NMD(a) CBG bioluminescence signal (lower panel) and ratios of CBR/CBG bioluminescence signals (upper panel) in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the indicated concentrations. The CBG signal and the CBR/CBG ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05; **P < 0.01 (paired t-test).(b) Western blot analysis of CBR-TCR(PTC) and CBG-TCR(WT) protein levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a.(c) Ratios of CBR-TCR(PTC)/CBG-TCR(WT) mRNA levels in U2OS reporter cells treated with DMSO or cardiac glycosides for 24 h at the concentrations indicated in a. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of four independent experiments. **P < 0.01; ***P < 0.001(paired t-test).(d) Effects of cardiac glycosides on the stability of the PTC-containing p53 mRNA in Calu-6 cells. Cells were treated with DMSO or cardiac glycosides at the concentrations indicated in a for 16 h before the addition of the transcriptional inhibitor actinomycin D (5 µg ml−1) to block new RNA synthesis. Total RNA was collected immediately before or 6 h after the addition of actinomycin D, and p53 mRNA levels were analyzed by RT-qPCR and normalized to GAPDH mRNA levels. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001(paired t-test).(e) Effects of ouabain on the stability of wild-type endogenous NMD target transcripts (UPP1, ATF-4, Pim3 and Pisd) in Calu-6 cells. ORCL mRNA, which is not a NMD target, was used as a control. Cells were treated with DMSO or ouabain, and then actinomycin D; samples were collected and analyzed as described in d. Data represent the percent mRNA remaining 6 h after transcriptional ablation (mean ± SD) from four independent experiments. **P < 0.01 (paired t-test).
Mentions: At the initial screening concentration (10 µM), all CGs increased CBR signal compared to DMSO treatment, but these drugs also decreased CBG signal, potentially due to non-specific toxic effects on cell viability (Supplementary Table 2). However, we identified a lower concentration for each drug at which the CBR activity was still dramatically increased while the CBG activity remained largely unaffected (Fig. 3a). At these modest concentrations, these drugs did not significantly impact general translation in cells and had only a mild inhibitory effect on cell viability (Supplementary Fig. 3a, b). Because CBR and CBG proteins are relatively stable, the inhibitory effects of CGs on the signal generated by the NMD reporter was time-dependent, increasing over the 24 h treatment period; CG-mediated blockade of NMD was fully concentration-dependent, showing reporter-generated EC50 values of ~70 nM and ~200 nM for ouabain and digoxin, respectively (Supplementary Fig. 4a, b). Importantly, our bioluminescence imaging results were corroborated by Western blot and reporter-specific RT-qPCR analyses of the CBR-TCR(PTC) and CBG-TCR(WT) protein and mRNA levels, respectively (Fig. 3b, c).

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