The unfolded protein response is shaped by the NMD pathway

Abstract

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD's ability to regulate normal mRNAs.

Keywords: ER stress; IRE1; NMD; UPR; cancer.

Figure 1. NMD targets several mRNAs encoding UPR components

1. qPCR analysis of HeLa cells stably depleted of UPF3A and UPF3B using shRNAs (as previously described 19) or transiently depleted of UPF1 using a siRNA. A value of 1 (dotted line) indicates expression in control HeLa cells transiently transfected with a luciferase siRNA or stably transfected with a shRNA luciferase construct (shLUC). mRNA levels were normalized against the mRNA encoding ribosomal protein L19, which is not affected by the UPR (n = 3).

2. Schematic representation of the Tet-off reporter, pTET2-βGlob-mini, containing the indicated sequences in the 3′ UTR region; β-292 is a NMD-resistant short 3′ UTR .

3. Pulse-chase mRNA half-life analysis of the indicated Tet-off reporter mRNAs. The reporter constructs were transiently transfected in HeLa Tet-off cells depleted of UPF1 (siUPF1) or luciferase (siLUC; control). Cells were treated with tetracycline to block transcription of the reporter promoter and samples were collected at the indicated time points. Values were normalized as in (A) (n = 6).

4. Pulse-chase mRNA half-life analysis of a Tet-off reporter mRNA harboring the full-length IRE1α 3′ UTR (β-IRE1α 3′UTR FL) versus the Tet-off reporter harboring the downstream and upstream deletions of the IRE1α 3′ UTR (β-IRE1α Del 1 and Del 2). Values were normalized as in (A) (n = 6).

5. Pulse-chase mRNA half-life analysis performed as in (D) in HeLa cells transiently depleted of UPF1 (siUPF1) or luciferase (siLUC; control) by RNAi. Values were normalized as in (A) (n = 3).

Data information: Statistical analysis by t-test (*P < 0.05). All error bars reflect SEM.

Figure 2. NMD raises the activation threshold of the UPR and promotes its attenuation

1. qPCR analysis of HeLa cells stably depleted of the NMD factor UPF3B (shUPF3B) and treated with increasing concentrations of tunicamycin (Tm) for 4 h. HeLa cells stably transfected with a construct expressing an shRNA against luciferase (shLUC) serves as a negative control (n = 6).

2. qPCR analysis of liver from Upf3b-null (n = 3) and matched control littermate (Wt) mice (n = 3) injected IP with the indicated concentrations of Tm for 12 h.

3. qPCR analysis of the stably transfected HeLa cells described in (A) treated with a low dose of Tm (0.25 μg/ml) for the times indicated (n = 6).

4. qPCR analysis of the stably transfected HeLa cells described in (A) and incubated with a high dose of Tm (2 μg/ml) for the time points indicated (n = 3).

5. qPCR analysis of liver from Upf3b-null (n = 3) and control littermate (Wt) mice (n = 3) injected IP with Tm (1 μg/g) for the time points indicated.

Data information: Values were normalized as in Fig1A and statistically analyzed by t-test (*P < 0.05). All error bars reflect SEM.

Figure 3. NMD suppresses UPR activation in response to endoplasmic reticulum stress

1. Western blot analysis of total IRE1α (top) and phospho-IRE1α (bottom) in HeLa cells stably depleted of the NMD factor UPF3B (shUPF3B) treated with a low dose of Tm (0.25 μg/ml) for the times indicated. HeLa cells stably transfected with a luciferase shRNA construct (shLUC) serve as a negative control. Phospho-IRE1α was detected by a mobility shift assay, as described . HSP-90 is the loading control, and the positive control is HEK293T cells treated with Tm (5 μg/ml) for 6 h.

2. RT–PCR analysis of XBP1 splicing under the same conditions as in (A) (representative of three independent experiments).

3. Western blot analysis of total ATF6 (top) and its cleavage product (ATF6-373) in cells under the same conditions as in (A).

4. Western blot analysis of CHOP and BIP in cells under the same conditions as in (A).

5. Quantification of cleaved ATF6 protein [ATF6 (373)] relative to the loading control, HSP90, from the Western blot data in (C).

Data information: Data in (A, C and D) are representative of two independent experiments.

Figure 4. NMD acts through IRE1α to shape the UPR

1. FACS analysis indicating the percentage of apoptotic (annexin-V positive/PI negative) HeLa cells in response to Tm (2 μg/ml) treatment for 48 h. HeLa cells were stably depleted of UPF3B (shUPF3B) or both UPF3A and UPF3B (shUPF3AB). HeLa cells stably transfected with a luciferase shRNA construct (shLUC) serve as a negative control. The values shown are the average (mean ± SEM) from three experiments relative to control (shLUC). The percentage of apoptotic cells in non-Tm-treated shLUC, shUPF3B, and shUPF3AB cells was 1.0, 1.8, and 2.1%, respectively (n = 3).

2. Quantification of apoptotic (TUNEL-positive) cells in liver tissue sections. Shown is the average number of TUNEL-positive cells in a field surrounding a hepatic portal area in Upf3b-null (n = 3) and control littermate (Wt) mice (n = 3) injected IP with Tm (1 μg/g) for the times indicated. Scoring was performed without knowledge of genotype.

3. qPCR analysis of HeLa cells (described in A) treated with a single low dose of tunicamycin (0.25 μg/ml) for the time points indicated (n = 3).

4. qPCR analysis of liver from Upf3b-null (n = 6) and control littermate (Wt) mice (n = 6).

5. qPCR analysis of the liver of Upf3b-null (n = 3) and control littermate mice (n = 3) injected IP with Tm (1 μg/g) for the times indicated.

6. qPCR analysis of the stably transfected HeLa cells described in (A) and transiently transfected with an IRE1α siRNA (siIRE1) or a control siRNA (siControl) and incubated with Tm (0.25 μg/ml) for 10 h (n = 3).

7. qPCR analysis of the stably transfected HeLa cells described in (A) that were incubated with IRE inhibitor STF-083010 (60 μM) and Tm (0.25 μg/ml) for 10 h (n = 3).

8. Model depicting the activity of the UPR and NMD pathways during the initiation (Init.), plateau (Plat.), and termination (Term.) phases of the UPR pathway.

9. Model depicting the NMD-UPR circuit defined by our data that amplifies the signal-to-noise ratio of endoplasmic reticulum stress responses.

Data information: Statistical analysis by t-test (*P < 0.05). In (C–G), values were normalized as in Fig1A. All error bars reflect SEM.