Intracellular calcium regulates nonsense-mediated mRNA decay

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.

Figure 1. A dual-color bioluminescence-based NMD reporter system

(a) Schematic diagram of the reporter construct containing two tandem, highly homologous transcription units, CBR-TCR(PTC) and CBG-TCR(WT). See Fig. S1 and text for details. (b–h) Validation of the NMD reporter depicted in a. (b) Western blot analysis of the CBR-TCR(PTC) and CBG-TCR(WT) protein levels in U2OS reporter cells treated with vehicle (H₂O) or caffeine. (c) Ratios of CBR/CBG bioluminescence signals in reporter cells treated with vehicle (H₂O) or caffeine. The ratio in H₂O-treated reporter cells was normalized to 1. Data represent the mean ± SD of four independent experiments. ***P < 0.001 (paired t-test). (d) Ratios of CBR-TCR(PTC)/CBG-TCR(WT) mRNA levels in reporter cells treated with vehicle (H₂O) or caffeine. The ratio in H₂O-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. ***P < 0.001 (paired t-test). (e) shRNA-mediated knockdown of the NMD factors SMG1 (2 shRNAs), UPF1 (1 shRNA) or UPF2 (2 shRNAs) in the dual-colored U2OS reporter cells. (f) Western blot analysis of CBR- TCR(PTC) and CBG-TCR(WT) protein levels in reporter cells after control-knockdown or SMG1-, UPF1-, or UPF2-knockdown. *, nonspecific band. (g) Ratios of CBR and CBG bioluminescence signals in reporter cells after control-knockdown or SMG1-, UPF1- or UPF2-knockdown. The ratio in control- knockdown reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates. ***P < 0.001; ****P < 0.0001 (t-test). (h) Ratios of CBR-TCR(PTC) and CBG-TCR(WT) mRNA levels in reporter cells after control-knockdown or SMG1-, UPF1- or UPF2-knockdown. The ratio in control-knockdown reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; *P < 0.05 (paired t-test).

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.

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).

Figure 4. Cardiac glycosides block NMD through inhibition of Na⁺/K⁺-ATPase and elevation of intracellular calcium

(a) Ratios of CBR/CBG bioluminescence signals in mouse dermal fibroblasts (left panel) or human U2OS cells (right panel) expressing the NMD reporter after 24 h treatment with DMSO, caffeine, or various concentrations of ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three biological replicates. (b) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit or rat α3 subunit of Na⁺/K⁺-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. *P < 0.05 (paired t-test). (c) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, human α1 subunit, or CG-resistant mutant human α1 subunit of Na⁺/K⁺-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test). (d) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells expressing either empty vector, rat α1 subunit, or catalytically-inactive rat α1 subunit of Na⁺/K⁺-ATPase after 24 h treatment with DMSO or ouabain. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test). (e) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 24 h treatment with DMSO, ouabain, ouabain and Bapta-AM, or Bapta-AM. Ouabain, 0.175 µM; Bapta-AM, 25 µM. The ratio in DMSO-treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01 (paired t-test). (f) Ratios of CBR/CBG bioluminescence signals in human U2OS reporter cells following 4 h treatment with DMSO, thapsigargin, thapsigargin and Bapta-AM, or Bapta-AM. Cells were pre-treated with either DMSO or Bapta-AM for 1 h before addition of thapsigargin. Thasipgargin, 0.2 µM; Bapta-AM, 50 µM. The ratio in DMSO- treated reporter cells was normalized to 1. Data represent the mean ± SD of three independent experiments. **P < 0.01; ***P < 0.001(paired t-test). (g) A model for the regulation of NMD by cardiac glycosides, Na⁺/K⁺-ATPase and intracellular calcium.