Induction of the Unfolded Protein Response at High Temperature in Saccharomyces cerevisiae

Abstract

Ire1 is an endoplasmic reticulum (ER)-located endoribonuclease that is activated in response to ER stress. In yeast Saccharomyces cerevisiae cells, Ire1 promotes HAC1-mRNA splicing to remove the intron sequence from the HAC1u mRNA ("u" stands for "uninduced"). The resulting mRNA, which is named HAC1i mRNA ("i" stands for "induced"), is then translated into a transcription factor that is involved in the unfolded protein response (UPR). In this study, we designed an oligonucleotide primer that specifically hybridizes to the exon-joint site of the HAC1i cDNA. This primer allowed us to perform real-time reverse transcription-PCR to quantify HAC1i mRNA abundance with high sensitivity. Using this method, we detected a minor induction of HAC1-mRNA splicing in yeast cells cultured at their maximum growth temperature of 39 °C. Based on our analyses of IRE1-gene mutant strains, we propose that when yeast cells are cultured at or near their maximum growth temperature, protein folding in the ER is disturbed, leading to a minor UPR induction that supports cellular growth.

Keywords: PCR; endoplasmic reticulum; mRNA solicing; yeast.

Figure 1

Oligonucleotide design for PCR primers that specifically amplify the HAC1i cDNA. (A) PCR primers used in this study. The red and green letters represent sequences hybridizing to the first and second HAC1 exon, respectively. (B) Through the oligo(dT)-primed RT reaction, total RNA samples obtained from DTT-treated (3 mM, 30 min) IRE1+ cells (YTH001), unstressed constitutive-UPR cells (YKY1002), and unstressed ire1Δ cells (YTH002) were converted to cDNA, which was then used as the template for PCR with the indicated primer sets. The PCR products were run on 2% agarose.

Figure 2

Quantitative measurement of the relative efficiency of HAC1-mRNA splicing through real-time PCR. (A) The cDNA sample generated by the oligo(dT)-primed RT reaction from the total RNA of constitutive-UPR cells (YKY1002) was 4-fold serially diluted and was subjected to real-time PCR in which the Forward-1/Reverse-5 primer set was used. (B) The cDNA sample generated by the oligo(dT)-primed RT reaction from total RNA of IRE1+ cells (YTH001) was 4-fold serially diluted and subjected to real-time PCR in which the Forward-2/Reverse-7 primer set was used. (C) The procedure for the real-time qPCR-based estimation of the relative HAC1 mRNA-splicing efficiency. (D) Amplification curves of the real-time PCR analysis. The oligo(dT)-primed cDNA samples were produced from RNA that was extracted from DTT-treated (3 mM, 30 min), unstressed IRE1+ cells (YTH001), and unstressed ire1Δ cells (YTH002). The primer sets are shown in panel C. (E) IRE1+ cells (YTH001) were stressed by the indicated concentrations of DTT for 30 min and checked for HAC1-mRNA splicing with the two different methods. The x-values were normalized against that of unstressed cells, which was set at 1.0.

Figure 3

UPR induction in cells grown at the upper-limit growth temperature of 39 °C. (A) and (B) after preculturing at 30 °C, the ire1Δ cells Y11907 transformed with the single-copy IRE1 plasmid pRS313-IRE1 (IRE1+) or the control empty vector plasmid pRS313 (ire1Δ) were shifted to 39 °C or were continuingly grown at 30 °C for monitoring of the relative HAC1 mRNA-splicing efficiency and the culture optical density. (C) The ire1Δ strain Y11907 transformed with pRS313-IRE1 (IRE1+) or its mutants (ΔIII or V535R) were incubated and analyzed as shown in panel A. To monitor the relative HAC1 mRNA-splicing efficiency, cells were harvested at the exponential growth phase, and the experimental procedure shown in Figure 2C was employed. Values in A and C were normalized against that of IRE1+ cells cultured at 30 °C, which were set at 1.0.