Attenuation of spliced HAC1 mRNA levels during the UPR. (A) ire1Δ yeast strain carrying WT IRE1 (Ire1-WT) was induced for ER stress with tunicamycin (Tm) at either middle (I) or early (II) log phases. Activation of IRE1 was monitored by HAC1 mRNA splicing on the Northern blots of isolated RNA from cells collected at various time points. Positions of spliced and unspliced HAC1 mRNA are indicated. Differences in HAC1 mRNA splicing are highlighted by the dotted blue boxes. (B) An extended UPR time course of Ire1-WT cells in the presence of Tm throughout the time course. The HAC1 mRNA membrane was reprobed for KAR2/BiP mRNA. (C) The percentage of HAC1 splicing (dotted line) over total HAC1 mRNA. (D) Ire1-WT cells continuously incubated with Tm were not able to splice HAC1 mRNA upon addition of 1 µg/ml of fresh Tm at the 8-h time point. (E) Tm in media that were incubated for 8 h was able to splice HAC1 in fresh cells that have never been treated with Tm. (F) The vacuolar carboxypeptidase Y (CPY) activity in Ire1-WT or ire1Δ cells with pRS341 was examined by the ability of CPY to cleave its substrate BTPNA to release p-nitroaniline, which was measured at 410 nm. The value of Ire1-WT cells before the UPR was set to 100% CPY activity, and the percentage of CPY activity in Ire1-WT or ire1Δ cells treated with Tm for the indicated lengths of time was calculated. (G) Levels of CPY activity for Ire1-WT cells were also shown (orange) and superimposed on the graph of HAC1 mRNA splicing from C. Error bars represent standard deviations calculated from at least three independent time courses.