mRNAs are down-regulated in response to multiple stressors in an Ire1-dependent manner. (A–C) mRNA abundance for targets of ER stress in hIre1^R and Ire1^−/− cells treated with 2 mM DTT (A), 3 µg/ml Tm (B), or 500 nM thapsigargin (C) for 5 h relative to untreated cells. (D) Relative mRNA abundance in cells lacking functional XBP-1. Cells were untransfected or transfected with negative control siRNA (QIAGEN) or siRNAs targeting Ire1, followed by DTT treatment as in A. (A–D) The means and SDs for three to four independent experiments were measured by qPCR; each sample was normalized using the mRNA levels of Rpl19.

mRNAs down-regulated by Ire1 are degraded faster in the presence of ER stress. hIre1^R, Ire1^−/−, or NIH-3T3 cells were treated with 1 µg/ml actinomycin D and/or 2 mM DTT, and relative mRNA abundance was monitored over time by qPCR. RNA levels were normalized to those of Rpl19 and to untreated controls. Error bars represent the SDs in qPCR replicates; representative time course data from two independent experiments are shown.

The nuclease activity of Ire1 is required for RIDD. (A) Western blot of Flag-tagged hIre1 proteins. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (B) Ethidium bromide–stained agarose gel showing XBP-1 reverse transcribed from total RNA and PCR amplified using primers surrounding the splice site. The asterisk denotes a hybrid band amplified from spliced and unspliced (u) products (Shang and Lehrman, 2004). s, spliced product after the removal of the 26 nucleotide intron. Bands were quantified in ImageJ (National Institutes of Health) to determine the percentage of splicing. (C and D) RNA levels of RIDD targets and controls in DTT (C)- and Tm (D)-treated cells measured by qPCR and normalized to signals from Rpl19. (B–D) The means and SDs for three independent experiments are shown.

Activation of Ire1 is not sufficient for RIDD. (A) Percentage of spliced XBP-1 in the absence (gray bars) and presence (black bars) of 7 µM 1NM-PP1 in the indicated cell lines. Splicing was measured as in Fig. 3 B. (B) Relative RNA abundance of ERdj4, measured by qPCR, in the absence (gray bars) and presence (black bars) of 1NM-PP1. RNA abundance was normalized to that of Rpl19, and ERdj4/Rpl19 ratios for experimental samples were divided by those for untreated cells from the same experiment. (C) Western blot of hIre-I642G–Flag. GAPDH, glyceraldehyde 3-phosphate dehydrogenase. (D and E) Relative RNA abundance of two RIDD targets, Blos1 (D) and Scara3 (E), measured as in B. Gray bars, no 1NM-PP1; black bars, 7 µM 1NM-PP1. (F) Relative RNA abundance in hIre1^R-I642G cells measured as in B. Control cells (shaded bars) and cells depleted of XBP-1 using RNAi (open bars) were treated with 1NM-PP1 only or 1NM-PP1 and DTT. (G and H) Percentage of spliced XBP-1 (G) and relative mRNA abundance for ERdj4 and Blos1 (H) in various concentrations of 1NM-PP1 and/or DTT. Measurements are as described for A and B. (A–H) Cells were treated with 2 mM DTT, 3 µg/ml Tm, and/or 7 µM 1NM-PP1 for 5 h unless otherwise indicated. The means and SD for three to five independent experiments are shown.