Hypoxic inhibition of nonsense-mediated RNA decay regulates gene expression and the integrated stress response

Abstract

Nonsense-mediated RNA decay (NMD) rapidly degrades both mutated mRNAs and nonmutated cellular mRNAs in what is thought to be a constitutive fashion. Here we demonstrate that NMD is inhibited in hypoxic cells and that this inhibition is dependent on phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha). eIF2alpha phosphorylation is known to promote translational and transcriptional up-regulation of genes important for the cellular response to stress. We show that the mRNAs of several of these stress-induced genes are NMD targets and that the repression of NMD stabilizes these mRNAs, thus demonstrating that the inhibition of NMD augments the cellular stress response. Furthermore, hypoxia-induced formation of cytoplasmic stress granules is also dependent on eIF2alpha phosphorylation, and components of the NMD pathway are relocalized to these granules in hypoxic cells, providing a potential mechanism for the hypoxic inhibition of NMD. Our demonstration that NMD is inhibited in hypoxic cells reveals that the regulation of NMD can dynamically alter gene expression and also establishes a novel mechanism for hypoxic gene regulation.

FIG. 1.

NMD is inhibited in hypoxic cells. (A) The stability of mRNA expressed from either wild-type β-globin (WT) or a β-globin cDNA with a premature termination codon (PTC 39) was determined in normoxic and hypoxic transiently transfected U2OS cells. Cells were rendered hypoxic or normoxic for 3 h, and then DRB was added during uninterrupted hypoxia. RNA was collected for analysis at time zero and then 4 h later. Normoxic and hypoxic β-globin expression at time zero was normalized to 1. The percentage of mRNA at each time point compared to time zero was determined, and the ratio of these percentages in hypoxic versus normoxic cells is depicted in the right panel; a ratio with a value of >1 indicates increased stability in hypoxic cells compared to normoxic cells for that time point. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments ± standard errors. (B) The mRNA and pre-mRNA expression levels of several NMD targets (DNAJB2, ARFRP1, MAP3K14, and UPP1) and control mRNA (ORC3L) were assessed in normoxic and in 3-h hypoxic U2OS cells by real-time PCR. Normoxic values were normalized to 1. Experiments were preformed in triplicate, and the graph depicts the averages ± standard errors. (C) U2OS cells were rendered hypoxic or normoxic for 3 h and then an inhibitor of RNA synthesis was added. RNA was collected for analysis at time zero and then 2, 4, and 6 h after RNA synthesis was inhibited, and the messages of NMD targets were determined by reverse transcription-PCR. Normoxic and hypoxic mRNA expression levels at time zero were normalized to 1. The hypoxic/normoxic ratio of mRNA levels at each time point is shown in the right panel, with values greater than 1 reflecting stability in hypoxic cells. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments + standard errors.

FIG. 2.

NMD inhibition in hypoxic cells is dependent on eIF2α phosphorylation. (A) The half-life of mRNA expressed from either wild-type β-globin (WT) or a β-globin cDNA with a premature termination codon (PTC 39) was determined in either normoxic or hypoxic stably transfected WT or eIF2α S51A MEFs. Cells were rendered hypoxic or maintained as normoxic for 3 h, at which time RNA was collected, and then cells were treated with DRB for 5 h and RNA was again collected. mRNA expression was normalized to 1. In the right panel mRNA stability is presented as a ratio, with a value of >1 indicating increased stability in hypoxic cells. Experiments were repeated in triplicate, and real-time PCR was performed in triplicate; graphs depict the averages ± standard errors. (B) The mRNA stabilities of several NMD targets were determined in normoxic or hypoxic wild-type MEFs or MEFs in which eIF2α cannot be phosphorylated (eIF2α S51A). Experiments were repeated in triplicate, and real-time PCR was performed in triplicate; graphs depict the averages + standard errors.

FIG. 3.

Stress response mRNAs are substrates of NMD. (A) Rent1/Upf1 was diminished with either siRNA pools or an shRNA lentivirus in U2OS and HeLa cells. Cellular stress was determined by assessing eIF2α phosphorylation by immunoblotting or XBP mRNA splicing by reverse transcription-PCR. Hypoxic stress (3 h) and tunicamycin (0.5 μg/ml for 3 h) served as positive controls. (B) U2OS cells were transiently transfected with either RFP or RFP-Rent1/Upf1 and harvested 48 h later for immunoblotting. eIF2α is shown as a loading control. (C) U2OS cells were transiently transfected with either RFP or RFP-Rent and 48 h later transfected again with 25% of the same plasmid and 75% of either a wild-type or PTC β-globin transgene. At 24 h, cells were harvested, RNA was isolated, and expression of β-globin was assessed by reverse transcription-PCR. Expression for each construct transfected with RFP only was normalized to 1. Real-time PCR was performed in triplicate, and the graphs depict the averages of two separate experiments ± standard errors. (D) Cells prepared as for panel B had total RNA extracted, and real-time PCR was performed to quantitate messages for ATF-4, ATF-3, and CHOP, transcripts degraded by NMD (DNAJB2, ARFRP1, and MAP3K14), and control messages not degraded by NMD (ORC3L, ID-1, and GAPDH). RNA expression at time zero was normalized to 1. Real-time PCR was preformed in triplicate, and the graphs depict the averages of two separate experiments ± standard errors. (E) mRNA stabilities of three cellular stress mRNAs were assessed in U2OS cells expressing either scrambled shRNA (scr) or Rent1/Upf1 shRNA (shRent) by inhibiting RNA synthesis with DRB and assessing mRNA expression at the time of inhibition and 2 and 8 hours later by real-time PCR. Expression for each message at time zero was normalized to 1. Real-time PCR was done in triplicate with two biological replicates.

FIG. 4.

eIF2α-dependent hypoxic inhibition of NMD stabilizes ATF-4, ATF-3, and CHOP to augment the stress response. (A) U2OS cells were rendered hypoxic or normoxic for 3 h prior to treatment with DRB. RNA was collected for analysis at time zero and then at 2, 4, and 6 h after addition of DRB (because of the short half-life of ATF-4, only time zero and 2 h were assessed), and the messages of NMD targets were determined by reverse transcription-PCR. Normoxic and hypoxic mRNA expression levels at time zero were normalized to 1. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments ± standard errors. The hypoxic/normoxic ratios of mRNA levels at each time point are shown in the right panel, with values greater than 1 reflecting stability in hypoxic cells. (B) Wild-type MEFs (eIF2α +/+) or MEFs which cannot phosphorylate eIF2α (eIF2α S51A) were treated as described for panel A, except the stability of ATF-4 mRNA was assessed after 1 h. (C) ATF-4^−/− MEFs were transfected with ATF-4 genomic constructs expressing either the coding region of ATF-4 or an ATF-4 cDNA containing the uORFs. The alternative uORFs are depicted by circles and hexagons. The region for ATF-4 coding is shown as a black rectangle, and introns are depicted as lines. Cells were rendered hypoxic for 3 h and then treated with an inhibitor of RNA synthesis. One hour after treatment, RNA was harvested and quantitated by real-time PCR. Normoxic cells were similarly treated. The endogenous ATF-4 half-life was determined with wild-type MEFs. mRNA expression in normoxic and hypoxic cells was normalized to 1 at time zero. Real-time PCR was preformed in triplicate, and the graphs depict the averages of four separate experiments ± standard errors. (D) U2OS cells were either infected with a lentivirus with a scrambled shRNA (scr) or a lentivirus with a Rent1/Upf1 sh RNA (shRent1/Upf1) as in Fig. 1A. Cells were then treated with 2.5 μg/ml of tunicamycin for short (A) or longer (B) times, protein was collected, and immunoblot assays were performed for proteins induced during the unfolded protein response (ATF-4, ATF-3, CHOP, and gadd34). Both actin and total eIF2α served as loading controls.

FIG. 5.

Hypoxic induction of stress granules is dependent on eIF2α phosphorylation, and Rent1/Upf1 protein is concentrated in these granules. A. WT MEFs, MEFs deficient for the PERK kinase, and MEFs which have knock-in alleles for eIF2α with a serine-to-alanine mutation so that eIF2α cannot be phosphorylated (S51A) were either rendered hypoxic for 3 h or treated for 3 h with 500 μM arsenic. Cells were then fixed and stained for a marker for stress granules (G3BP). B. Cos-1 cells were transiently transfected with a Dcp1a-RFP construct and 24 h later plated on coverslips. Then, 24 h later they were either treated with 500 μM arsenic or rendered hypoxic for 3 h or untreated. Cells were then fixed and stained for antibodies directed against components of either stress granules (G3BP) or processing bodies (Ge-1/Hedls, which cross-reacts with the p70 S6 kinase antibody). C. Cos-1 cells were transiently transfected with RFP-Rent1/Upf1. Cells were then rendered hypoxic for 3 hours or treated with arsenic, and stress granules were visualized with indirect immunofluorescence.

FIG. 6.

Hypothesis for hypoxic suppression of NMD via sequestration of NMD targets in stress granules. In this hypothetical model, cellular hypoxia induces stress granule formation, which is dependent on eIF2α phosphorylation, and leads to the sequestration of Rent1/Upf1 and Rent1/Upf1-tethered NMD targets in stress granules. Stress granules, as opposed to processing bodies, do not contain the enzymes necessary to degrade NMD. Therefore, these Rent1/Upf1-tethered mRNAs are stabilized.