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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 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).
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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).

Mentions: To investigate the NMD pathway and to begin to develop NMD-targeting therapeutics, we constructed a multicolored, bioluminescence-based reporter for assaying NMD in mammalian cells, as illustrated in Fig. 1a and Supplementary Fig. 1. This reporter comprises a single expression vector containing two separate transcription units, each with a luciferase inserted into a TCRβ minigene at the same position within the second exon. The first transcription unit consists of a PTC-containing TCRβ minigene fused to click beetle red luciferase (CBR-TCR(PTC)). The second unit contains a wild-type TCRβ minigene fused to click beetle green 99 luciferase (CBG99, hereafter referred to as CBG for simplicity) (CBG-TCR(WT)). Expression of both fusion reporter genes are controlled by separate CMV promoters, splice sites, and polyadenylation signals of identical sequences. A sequence encoding an HA-tag was included in the first exon of the fusion reporter genes, which provides an independent method to detect the translated fusion protein products through Western blotting. PTCs in the well characterized TCRβ minigene are known to elicit robust NMD (but not 100% efficient as is the case for other reporter genes examined)10,11. The CBR-TCR(PTC) and CBG-TCR(WT) transcription units share > 99% sequence identity at the DNA, pre-mRNA, and mRNA levels (see the reporter sequence in Supplementary Fig. 2). Using this dual-colored reporter, NMD is quantified by the ratio of CBR activity to CBG activity, with an increase in the CBR/CBG (red/green) ratio representing inhibition of NMD. Here, the CBR luciferase activity serves as an indirect measure of the steady-state levels of the CBR-TCR(PTC) fusion mRNA, which is targeted for degradation by NMD, whereas the CBG luciferase activity reflects the steady-state levels of the CBG-TCR(WT) fusion mRNA, which is unresponsive to NMD. The use of CBG-TCR(WT) as an internal control in the same cell ensures that changes in the CBR/CBG ratio reflect effects specifically attributable to NMD, but not indirect effects that result from variations in reporter DNA delivery or from effects on cell viability or various steps of gene expression such as transcription, splicing, polyadenylation, and translation. The use of the highly sensitive and closely related red-emitting CBR and green-emitting CBG luciferases, combined with a spectral deconvolution algorithm for unmixing CBR and CBG signals, allows rapid and accurate measurement of their respective activities simultaneously in a single reaction with the same D-luciferin substrate12.


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 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).
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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).
Mentions: To investigate the NMD pathway and to begin to develop NMD-targeting therapeutics, we constructed a multicolored, bioluminescence-based reporter for assaying NMD in mammalian cells, as illustrated in Fig. 1a and Supplementary Fig. 1. This reporter comprises a single expression vector containing two separate transcription units, each with a luciferase inserted into a TCRβ minigene at the same position within the second exon. The first transcription unit consists of a PTC-containing TCRβ minigene fused to click beetle red luciferase (CBR-TCR(PTC)). The second unit contains a wild-type TCRβ minigene fused to click beetle green 99 luciferase (CBG99, hereafter referred to as CBG for simplicity) (CBG-TCR(WT)). Expression of both fusion reporter genes are controlled by separate CMV promoters, splice sites, and polyadenylation signals of identical sequences. A sequence encoding an HA-tag was included in the first exon of the fusion reporter genes, which provides an independent method to detect the translated fusion protein products through Western blotting. PTCs in the well characterized TCRβ minigene are known to elicit robust NMD (but not 100% efficient as is the case for other reporter genes examined)10,11. The CBR-TCR(PTC) and CBG-TCR(WT) transcription units share > 99% sequence identity at the DNA, pre-mRNA, and mRNA levels (see the reporter sequence in Supplementary Fig. 2). Using this dual-colored reporter, NMD is quantified by the ratio of CBR activity to CBG activity, with an increase in the CBR/CBG (red/green) ratio representing inhibition of NMD. Here, the CBR luciferase activity serves as an indirect measure of the steady-state levels of the CBR-TCR(PTC) fusion mRNA, which is targeted for degradation by NMD, whereas the CBG luciferase activity reflects the steady-state levels of the CBG-TCR(WT) fusion mRNA, which is unresponsive to NMD. The use of CBG-TCR(WT) as an internal control in the same cell ensures that changes in the CBR/CBG ratio reflect effects specifically attributable to NMD, but not indirect effects that result from variations in reporter DNA delivery or from effects on cell viability or various steps of gene expression such as transcription, splicing, polyadenylation, and translation. The use of the highly sensitive and closely related red-emitting CBR and green-emitting CBG luciferases, combined with a spectral deconvolution algorithm for unmixing CBR and CBG signals, allows rapid and accurate measurement of their respective activities simultaneously in a single reaction with the same D-luciferin substrate12.

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