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1 HSP Research Institute, Kyoto 600-8813, Japan
2 Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan
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Abstract |
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 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
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Introduction |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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It is well established that the expression and activity of Hac1p are tightly regulated at the level of mRNA splicing that depends on an unconventional system. HAC1 mRNA is constitutively synthesized as an intron-containing precursor mRNA under normal conditions and spliced in response to ER stress (Cox & Walter 1996; Kawahara et al. 1997). Splicing of HAC1 precursor mRNA is initiated by Ire1p-mediated cleavage (Sidrauski & Walter 1997); Ire1p is a transmembrane protein kinase/endoribonuclease localized in the ER that is activated by ER stress-induced oligomerization and autophosphorylation (Cox et al. 1993; Mori et al. 1993; Shamu & Walter 1996; Welihinda & Kaufman 1996). Cleaved HAC1 mRNA is ligated by the action of Rlg1p, a tRNA ligase, to produce mature mRNA (Sidrauski et al. 1996). Splicing regulates the synthesis of active Hac1p at two levels. First, Hac1p is synthesized only after the splicing occurs since the HAC1 intron blocks mRNA translation (Chapman & Walter 1997; Kawahara et al. 1997). Second, the transcriptional activator activity of Hac1p translated from spliced (mature) mRNA is much stronger than that of Hac1p translated from unspliced (precursor) mRNA since the DNA-binding domain encoded by the first exon is joined with the activation domain encoded by the second exon as a result of mRNA splicing (Mori et al. 2000) (see Discussion for details). Hac1p thus synthesized potently activates transcription in the nucleus via direct binding to the cis-acting UPR element (UPRE) present in the promoter regions of UPR-target genes (Mori et al. 1992, 1998). This unique and tight regulation allows yeast cells to produce a highly active transcription factor Hac1p only when cells need transcriptional induction of UPR-target genes to cope with unfolded proteins accumulated in the ER.
In this report, we show that the HAC1 promoter itself carries a functional UPRE to which Hac1p can directly bind. Thus, once produced via mRNA splicing, Hac1p can up-regulate its own transcription as long as Hac1p is synthesized under ER stress conditions. Importantly, this autoregulation is required to maintain high levels of HAC1 mRNA and thereby protect yeast cells from prolonged ER stress.
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Results |
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An approximately 1 kb fragment immediately upstream of the HAC1 coding sequence was fused in-frame to the Escherichia coli lacZ gene to test whether the HAC1 promoter responds to ER stress to activate transcription of its downstream gene. As shown in Fig. 1, the lacZ mRNA level was markedly enhanced in yeast cells expressing the HAC1 promoter-lacZ fusion gene 30 min after tunicamycin treatment; tunicamycin evokes ER stress by inhibiting protein N-glycosylation (Kaufman 1999). Accordingly, the ß-galactosidase level expressed inside the cells increased 2- to 3-fold after 3 h incubation with tunicamycin. These induction patterns observed for the HAC1 promoter at the mRNA and protein levels were almost indistinguishable from those for the KAR2 promoter (Fig. 1, compare lanes 3 and 4 with lanes 5 and 6); the KAR2 gene encodes an ER-localized molecular chaperone of the HSP70 family and represents a major target of the UPR (Normington et al. 1989; Rose et al. 1989), indicating that HAC1 is an ER stress-inducible gene. It appears that such a clear notion has been masked by the splicing event that occurs in response to ER stress.
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Next, various 5' deletion mutants of the HAC1 promoter were created, fused in-frame to the lacZ gene, and then analysed for their ability to respond to ER stress. The results of ß-galactosidase assays shown in Fig. 2A,B indicated that the region –200 to –240 (the adenine of the first ATG codon is set as +1) was critically involved in the induction. Then, various regions of 30 nucleotides between –181 and –260 (see Fig. 3A) were inserted into the upstream region of the CYC1 promoter-lacZ fusion gene, which alone does not respond to ER stress (Fig. 3B, line 7). Insertion of the region D (–201 to –230) made the CYC1-lacZ fusion gene highly inducible by ER stress (Fig. 3B, line 4) to an extent very similar to that of the UPRE present in the KAR2 promoter (KAR2 UPRE, Fig. 3B, line 8). These results indicated that the HAC1 region D is necessary and sufficient for the induction by ER stress.
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) or Hac1p (hac1) as in the case of KAR2 UPRE (Fig. 3C), suggesting that the HAC1 region D may contain a functional UPRE. Our previous extensive mutagenic analyses on KAR2 UPRE and other UPREs have revealed that a palindromic sequence separated by a spacer of one nucleotide C (CANCNTG) is an essential element of the UPRE (Mori et al. 1996, 1998). Such a characteristic sequence was found in the HAC1 region D, which was centred at nucleotide –212 (CACCTTG) (Fig. 4). The importance of this ‘palindromic core sequence’ in the function of the HAC1 region D was demonstrated by ß-galactosidase assays, in which three adjacent nucleotides in the HAC1 region D were simultaneously mutated by transversion and their effects on the response to ER stress were determined (Fig. 4, lines 7–9). We also showed previously that the longer the palindrome in an UPRE-like sequence, the stronger is its transcriptional activity (Mori et al. 1996). Consistent with this notion, 5 nucleotides are completely palindromic (GAACA vs. TGTTC) in the HAC1 region D and replacement of the nucleotides outside of the palindromic core sequence also had deleterious effects on the transcriptional activity of the HAC1 region D (Fig. 4, lines 6 and 10). As these results indicated that the HAC1 region D conformed to our rules for a functional UPRE, we refer to the HAC1 region D as HAC1 UPRE hereafter.
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To examine whether HAC1 UPRE constitutes a binding site for Hac1p similarly to KAR2 UPRE, electrophoretic mobility shift assays (EMSA) were carried out using whole cell extracts prepared from the ERN+ and hac1 strains. As reported previously (Kawahara et al. 1997), cellular binding activity to 32P-labelled KAR2 UPRE was detected only after the ERN+ strain was ER-stressed (Fig. 5A, lanes 1 and 2) and was not detected in the hac1 strain before or after ER stress treatment (Fig. 5A, lanes 3 and 4). Similarly, cellular binding activity to 32P-labelled HAC1 UPRE was detected after ER stress treatment of the ERN+strain but not of the hac1 strain (Fig. 5A, lanes 5–8). This cellular binding to 32P-labelled HAC1 UPRE (Fig. 5B, lane 1) was competed by unlabelled HAC1 UPRE in a dose-dependent manner (Fig. 5B, lanes 2 and 3) and by unlabelled KAR2 UPRE with similar efficiency (Fig. 5B, lanes 6 and 7), indicating that the same cellular factor or factors bind to both HAC1 UPRE and KAR2 UPRE in a Hac1p-dependent manner. Importantly, this Hac1p-dependent cellular binding to 32P-labelled HAC1 UPRE (Fig. 5B, lane 1) was not competed by a mutant version of HAC1 UPRE designated M6 (Fig. 5B, lanes 4 and 5), which lost the ability to respond to ER stress due to the mutation of the three nucleotides in the palindrome core sequence essential for the UPRE activity (see Fig. 4, line 7), suggesting that transcriptional activity of HAC1 UPRE correlated well with its binding activity to Hac1p.
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Autoregulation of the HAC1 gene is required for sustained expression of HAC1 mRNA and sustained resistance to ER stress
Since the mutation of the palindromic core sequence in HAC1 UPRE from CACCTTG to gtgCTTG (three mutated nucleotides are in small letters) abolished both its response to ER stress (Fig. 4, line 7) and its binding to Hac1p (Fig. 5B, lanes 4 and 5), we mutated the three nucleotides in the context of the HAC1 promoter and examined the effects on promoter activity. As shown in Fig. 6A, the wild-type HAC1 promoter fused to the lacZ gene responded well to ER stress in the ERN+ but not in ire1 or hac1 strain by inducing ß-galactosidase activity, as expected. In contrast, the mutant HAC1 promoter carrying 3 point mutations failed to do so regardless of the cell type employed when similarly fused to the lacZ gene, indicating that HAC1 UPRE is essential for autoregulation of the HAC1 gene.
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strain. As shown in Fig. 6B, the mutation did not significantly affect either the level of HAC1 precursor (unspliced) mRNA constitutively expressed in the transformants (time 0) or ER stress-induced splicing of HAC1 mRNA that was observed within 30 min after tunicamycin treatment (time 0.5), indicating that the mutation in HAC1 UPRE per se had no impact on the expression of Hac1p under normal and short ER stress conditions. Importantly, however, when cells were subjected to prolonged ER stress, high levels of unspliced and spliced HAC1 mRNA were maintained even after 7.5 h treatment with tunicamycin in cells expressing the HAC1 gene from the wild-type promoter, whereas the levels of both unspliced and spliced HAC1 mRNA decreased considerably under the same conditions when the HAC1 gene was expressed from the mutant promoter. Thus, autoregulation of the HAC1 genes is required to maintain high level expression of HAC1 mRNA in a sustained fashion.
We finally examined whether autoregulation of the HAC1 gene is important for the ability of yeast cells to survive under ER stress conditions. For this purpose, we took advantage of the sec53 mutant, a yeast strain having a temperature-sensitive defect in phosphomannomutase (Feldman et al. 1987). By simply raising the temperature from 23 °C (permissive) to 30 °C (semi-nonpermissive), the sec53 mutant accumulates full-length precursors of secretory proteins which are abnormally glycosylated and malfolded in the ER (Feldman et al. 1987), thus causing ER stress (Normington et al. 1989; Rose et al. 1989). We have already reported that the sec53, hac1 strain rapidly loses viability at 30 °C due to the lack of cellular UPR activity, whereas the sec53, HAC1 strain is resistant to such ER stress-induced cell death (Mori et al. 1996). These observations were well reproduced in Fig. 6C. Most importantly, the sec53, hac1 strain expressing the HAC1 gene from the wild-type promoter behaved like the sec53, ERN+ strain and showed an approximately 50% survival rate even 12 h after the temperature shift to 30 °C, whereas the sec53, hac1 strain expressing the HAC1 gene from the mutant promoter gradually lost viability from 1 h after the temperature shift and exhibited only a 1% survival rate after 12 h. These results clearly indicated that autoregulation of the HAC1 gene is required to protect cells against prolonged ER stress.
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Discussion |
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Here, we explored a new key regulation mechanism for Hac1p: Hac1p activates its own transcription by directly binding to the cis-acting UPRE present in the HAC1 promoter, which is necessary and sufficient for the induction of the HAC1 gene by ER stress. Cells devoid of this autoregulation cannot maintain high levels of HAC1 mRNA and thereby cannot survive very well under prolonged ER stress conditions. We therefore propose that this regulation at the transcription level is required for sustained activation of the UPR.
No gene homologous to the yeast HAC1 gene can be identified in the genome of Caenorhabditis elegans, mouse or humans. Instead, its functional homolog has recently been identified as the XBP1 gene in both the worm and mammals although they are dissimilar in sequence (Shen et al. 2001; Yoshida et al. 2001; Calfon et al. 2002; Lee et al. 2002). Transcriptional induction of ER chaperone genes by ER stress in mammals is mediated by the cis-acting ER stress response element (ERSE) present in their promoter regions (Yoshida et al. 1998; Roy & Lee 1999). The consensus sequence of ERSE is CCAAT-N9-CCACG and thus quite different from the consensus sequence of yeast UPRE (CANCNTG). As the general transcription factor NF-Y constitutively occupies the CCAAT part of ERSE (Li et al. 1994), mammalian UPR-specific transcription factor has been expected to bind to the CCACG part of ERSE. XBP1 was isolated as a bZIP protein that binds to the CCACG part of ERSE using yeast one-hybrid screening (Yoshida et al. 1998). Subsequently, it turned out that both worm and mammalian XBP1 mRNAs are substrates of the ER stress-induced unconventional splicing system, which relies on the worm or mammalian homolog of yeast Ire1p (Shen et al. 2001; Yoshida et al. 2001; Calfon et al. 2002; Lee et al. 2002). The splicing event replaces the C-terminal region of XBP1 without affecting the N-terminal region containing the bZIP domain as in the case of Hac1p. Since the new C-terminal region functions as a potent activation domain, the DNA-binding domain and activation domain of XBP1 are joined as a result of mRNA splicing to produce a highly active transcription factor XBP1 (Yoshida et al. 2001). Thus, the activation mechanism of Hac1p and XBP1 is well conserved. Based on these findings, we proposed to term this novel mechanism ‘frame switch splicing’ to discriminate it from spliceosome-mediated conventional splicing (alternative splicing), wherein an open reading frame is switched by the presence or absence of mRNA splicing (Yoshida et al. 2001, 2003; Mori 2003). Ire1p-mediated frame switch splicing can remove an intron from mature mRNA which has been produced by alternative splicing and confer a new property on the protein encoded by the target mRNA by changing its C-terminal region without affecting its N-terminal region.
The HAC1 intron consisting of 252 nucleotides strongly inhibits mRNA translation by forming base-pairing with the 5' untranslated region of HAC1 mRNA and by being stalled at the ribosomes (Ruegsegger et al. 2001). In contrast, the worm or mammalian XBP1 intron consisting of only 23 or 26 nucleotides, respectively, is too short to form such inhibitory base-pairing (Yoshida et al. 2001; Calfon et al. 2002). Therefore, XBP1 mRNA constitutively expressed at a low level is translated even in the absence of splicing. It should be noted that XBP1 produced at a low level from unspliced mRNA is unable to activate transcription due to its low transcriptional activator activity as well as rapid degradation by the proteasome. Interestingly and importantly, XBP1 mRNA is inducible by ER stress at least in mammals and its induction time course is similar to that of ER chaperones (Yoshida et al. 2000). Consistent with this notion, the XBP1 promoter carries a functional ERSE (Yoshida et al. 2000), allowing XBP1 to activate its own transcription. XBP1 produced at a high level from induced and spliced mRNA possesses higher transcriptional activity and can escape from proteasome-mediated degradation, leading to transcriptional induction of its target genes.
Our results described here indicate that autoregulation is another feature of substrates of frame switch splicing that is conserved from yeast to humans in addition to the C-terminal replacement. Due to this autoregulation, both yeast and mammalian cells can continue synthesizing the UPR-specific transcription factors Hac1p and XBP1, respectively, to cope with unfolded proteins accumulated in the ER and can survive even under prolonged ER stress conditions. It should be emphasized that this autoregulatory mechanism does not harm the shut-off mechanism at least in the case of yeast UPR. When unfolded proteins are refolded by the action of induced ER chaperones or cleared by degradation via the ER-associated degradation system, the ER stress sensor molecule Ire1p returns to the inactive monomer via re association with the ER chaperone BiP/Kar2p (Bertolotti et al. 2000; Okamura et al. 2000), so that no more frame switch splicing occurs in recovered cells. Once the splicing reaction stops, newly synthesized HAC1 mRNA is no longer translated due to the presence of an intron, and preexisting Hac1p disappears rapidly in the cells because of its short half-life of 2 min (Chapman & Walter 1997; Kawahara et al. 1997). Thus, yeast cells can shut off the UPR immediately, whenever unfolded proteins in the ER are dismissed, even after they have undergone with prolonged ER stress. We conclude that yeast cells have developed a sophisticated way to cope with unfolded proteins accumulated in the ER.
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Experimental procedures |
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TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
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The yeast strains used in this study were KMY1005 (MAT
leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801), KMY1015 (KMY1005 ire1::TRP1), KMY1045 (KMY1005 hac1::TRP1), KMY2005 (MAT leu2-3,112 ura3-52 his3-200 trp1-901 lys2-801 sec53-6) and KMY2045 (KMY2005 hac1::TRP1) (Mori et al. 1996). The compositions of rich broth medium (YPD) and synthetic complete medium used for the selection of transformants such as SC(-Ura) have been previously described (Sherman et al. 1986). Tunicamycin (T-7765) was obtained from Sigma (St. Louis, MO) and used at a concentration of 5 µg/mL throughout the experiments. Yeast cells were transformed by the lithium acetate method (Ito et al. 1983).
Construction of plasmids
Recombinant DNA techniques were carried out as previously described (Sambrook et al. 1989). A BamHI site was created immediately downstream of the start ATG codon (ATGGATCC-) of the HAC1 gene by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA, USA). An approximately 1 kb BamHI fragment encompassing the HAC1 promoter region was inserted into the BamHI site of pSEYc102 (a CEN4-ARS1-based single-copy vector containing the URA3 selectable marker) (Mori et al. 1992). In the resulting plasmid, which was used for the experiments in Figs 1 and 2, the start ATG codon was in-frame to the lacZ coding sequence present in pSEYc102. Various 5' deletion mutants of the HAC1 promoter were created using appropriate restriction enzyme sites (Fig. 2A) or by site-directed mutagenesis (Fig. 2B), and then inserted between the SmaI and BamHI sites of pSEYc102. pSEYc102 carrying the 0.3 kb wild-type KAR2 promoter-lacZ fusion gene was constructed previously (Mori et al. 1992).
Double-stranded oligonucleotides corresponding to various subregions of the HAC1 promoter were inserted between the EcoRI and XhoI sites (immediately upstream of the CYC1-lacZ fusion gene) of pMCZ2 (a 2 µm-based multicopy vector containing the URA3 selectable marker) (Mori et al. 1998) to be used for the experiments in Fig. 3. A double-stranded oligonucleotide encoding the HAC1 region D or its various mutants was also inserted between the EcoRI and XhoI sites of pMCZ2 to be used for the experiments in Fig. 4.
The wild-type or mutant (M6) version of the 0.3 kb Bpu1102I-BamHI fragment of the HAC1 promoter was inserted between the SmaI and BamHI sites of pSEYc102 to create YCp-PHAC1(WT)-lacZ or YCp-PHAC1(mutant)-lacZ, respectively, which were used for the experiments in Fig. 6A. The wild-type or mutant (M6) version of the 0.3 kb Bpu1102I-BamHI fragment of the HAC1 promoter was also fused in-frame to the HAC1 coding sequence in YCp-L2 (a CEN4-ARS1-based single-copy vector containing the LEU2 selectable marker) (Mori et al. 1996) to create YCp-PHAC1(WT)-HAC1 or YCp-PHAC1(mutant)-HAC1, respectively, which were used for the experiments in Fig. 6B,C.
Assays
ß-Galactosidase assays and Northern blot hybridization analysis were carried out as previously described (Mori et al. 1996; Kawahara et al. 1997). EMSA was performed as previously described (Mori et al. 1996). Cell extracts were prepared from the wild-type (KMY1005) and hac1 (KMY1045) strains that had been grown in YPD medium to a mid-log phase and incubated for 1 h with tunicamycin, and proteins were fractionated by ammonium sulphate according to our previous report (Kawahara et al. 1997). Hac1p was translated in vitro using the TNT-Coupled Wheat Germ Extract System (Promega, Madison, WI) and a template HAC1 DNA according to the manufacturer's instructions. Hac1p fused to GST and Hac1p fused to MBP were expressed and purified from bacterial cells as previously described (Mori et al. 1996).
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Acknowledgements |
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Footnotes |
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* Correspondence: Email: kazu.mori{at}bio.mbox.media.kyoto-u.ac.jp
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References |
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Received: 10 October 2003
Accepted: 17 November 2003
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, respectively). KMY2045 and the sec53, HAC1 strain KMY2005 were also transformed with YCp-L2 alone (marked by •and 
, respectively). Transformants were grown at 23 °C in SC(-Leu) medium to an early mid-log phase, and then shifted to 30 °C for the indicated periods. The survival of cells was measured by plating appropriate dilutions on YPD plates. Values are expressed as a percentage of the control.