IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor.
In eukaryotic cells, proteins synthesised in the endoplasmic reticulum (ER) are appropriately folded and assembled before leaving the ER. If these processes are disturbed, unfolded proteins accumulate in the ER and the signalling pathway from the ER to the nucleus is activated to induce the transcription of genes encoding the ER protein quality control molecules, such as molecular chaperones, folding enzymes and components of ER-associated protein degradation as well as other stress related proteins. This cellular response conserved in eukaryotes is referred to as the unfolded protein response or the ER stress response1,2.
IRE1 plays an essential role in the signalling pathway of the ER stress response in yeast. IRE1 is an ER-resident type I transmembrane protein with a sensor domain in its luminal side and kinase and RNase domains in its cytoplasmic side. When sensing ER stress, IRE1 oligomerises and transactivates its RNase activity by autophosphorylation through its kinase activity3. Dissociation of BiP from the sensor domain triggers this oligomerisation4, whereas the sensor domain itself is considered to recognise unfolded proteins5. The activated RNase domain cleaves 2 specific phosphodiester bonds in HAC1 mRNA that encodes a bZIP transcription factor6,7. After cleavage, 5′ and 3′ fragments of HAC1 mRNA are joined by the tRNA ligase Rlg18. Splicing removes 252 nucleotides from the precursor mRNA HAC1u to generate the spliced HAC1 mRNA HAC1s. HAC1s encodes HAC1s protein consisting of 238 amino acids, which is larger than the HAC1u protein (230 amino acids). The HAC1s protein contains an 18-amino acid sequence that is important for transcription factor activity, which is missing in the HAC1u protein9.
Two IRE1 homologues, IRE1α and IRE1β, have been indentified in mammalian cells. Mammalian IRE1α is considered to be a functional homologue of yeast IRE1 because IRE1α catalyses unconventional splicing of XBP1 mRNA in a manner analogous to its yeast counterpart10. In this case, splicing removes a 26-nucleotide segment, which is much shorter than the HAC1 mRNA intron. Nevertheless, yeast HAC1 and human XBP1 share a secondary structural feature around the splicing sites i.e. both HAC1 and XBP1 mRNAs have 2 stem-loop structures with conserved ribonucleotides in the 7-nucleotide loops10,11. The precursor XBP1 mRNA (XBP1u) encodes an XBP1u protein of 261 amino acids; however, because of a frameshift caused by splicing, the spliced XBP1 mRNA (XBP1s) produces the XBP1s protein of 376 amino acids with a different and longer amino acid sequence at the C-terminus. As in the case of the HAC1s protein, the XBP1s protein gains a transcriptional activator domain in the new C-terminal region10,11. Mammalian cells have 2 additional ER stress sensors: the membrane-bound transcription factor ATF6 and the receptor-type protein kinase PERK. ATF6 is anchored to the ER membrane by virtue of its transmembrane domain (TMD) and is activated by proteolysis mediated by the proteases S1P and S2P12,13.
In plants, 2 IRE1 homologues, IRE1A and IRE1B, have been reported in Arabidopsis (Arabidopsis thaliana)14,15. Both IRE1A and IRE1B are localised in the ER and their putative sensor domains function as an actual sensor in yeast15. However, their involvement in the ER stress response remains to be determined. Similar to yeast and mammalian cells, Arabidopsis IRE1A and IRE1B may also catalyse unconventional splicing of a certain mRNA: however, such an mRNA is yet to be identified.
Arabidopsis also has stress transducers similar to those of mammals. bZIP17 and bZIP28 have a TMD, as does ATF6 and are therefore considered to be activated by proteolysis mediated by the proteases S1P and S2P16,17,18. bZIP60 was identified as a bZIP transcription factor whose transcript is induced by treatment with tunicamycin, an inhibitor of N-linked glycosylation widely used to induce the ER stress response19. bZIP60 encodes 295-amino acid protein that consists of a transcriptional activation domain, bZIP domain, putative TMD and C-terminal region19,20. Induction of several tunicamycin-inducible genes was reduced in the bzip60 mutant, providing evidence that bZIP60 is involved in transcriptional activation of ER stress-inducible genes21.
Several experiments were performed to elucidate the underlying mechanism of bZIP60 activation. First, a transient expression assay showed that expression of bZIP60ΔC, an artificial truncated form of bZIP60 whose TMD and C-terminal region were deleted, activated promoters of ER chaperone genes, whereas expression of the native form of bZIP60 did not19. Second, an immunoblot analysis using an antibody raised against bZIP60ΔC showed that in addition to the native form of bZIP60, a faster-migrating, smaller form of bZIP60 existed and was present only when plants were treated with tunicamycin21. Third, a subcellular fractionation experiment showed that the native and smaller forms of bZIP60 were localised to the ER and the nucleus, respectively21. On the basis of these observations, it was hypothesized that the bZIP60 protein is bound to the ER membrane through its TMD and is cleaved in response to ER stress, thereby producing the nuclear-localised, active bZIP6021,22. However, the molecular basis of bZIP60 activation has been enigmatic because neither S1P nor S2P appear to be involved in the production of the active form of bZIP60 protein21. In the current study, we present evidence that the active form of bZIP60 protein is synthesised from mRNA spliced by IRE1A and IRE1B.
A double mutant of IRE1 homologues exhibited tunicamycin sensitivity
T-DNA insertion mutants of Arabidopsis IRE1A (ire1a) and IRE1B (ire1b) were obtained from stock centres of T-DNA insertion lines (Fig. 1a). We generated ire1a and ire1b single mutants as well as an ire1a/ire1b double mutant (Fig. 1b and Supplementary Fig. S1 online, see Methods section for nomenclature of IRE1 genes and their mutants). All of these mutants were visibly indistinguishable from wild-type plants under ambient laboratory conditions grown on soil. However, when their seeds were sown on medium containing various concentrations of tunicamycin, germination of the ire1a/ire1b double mutant was more severely inhibited than that of the wild-type and single mutants (Fig. 1c, d and Supplementary Fig. S2 online), implying the involvement of both IRE1A and IRE1B in the ER stress response.
Differences in sensitivity against tunicamycin might be due to a decreased transcriptional induction of a gene encoding UDP-N-acetylglucosamine:dolichol phosphate N-acetylglucosamine-1-P transferase (GPT), an enzyme that catalyses the initial step of the N-linked glycan biosynthetic pathway and whose activity is inhibited by tunicamycin. This inference is based on the fact that in our previous study, an increased expression of GPT conferred resistance to tunicamycin23. Therefore we analysed GPT expression in ire1 mutants. Induction of GPT was not affected in any of the ire1 mutants (Supplementary Fig. S3 online).
Genes whose induction was reduced in ire1a/ire1b largely overlapped those in bzip60
To investigate how ER stress-inducible genes are affected in the ire1a/ire1b double mutant, seedlings of the wild-type and ire1a/ire1b were mock or tunicamycin treated and subjected to transcriptome analysis using a microarray representing approximately 24,000 genes in Arabidopsis. A hybridisation signal of 162 genes was increased 2.5-fold or more at 5 h of tunicamycin treatment in the wild-type with P value of less than 0.05 (see Methods for details) (Supplementary Table S1 online). Of these, 59 genes showed 50% or less induction by tunicamycin treatment in ire1a/ire1b than that in the wild-type, indicating that IRE1s are involved in the induction of the ER stress-inducible genes, although we could not exclude the possibility that the difference in the level of transcript accumulation between wild-type and ire1a/ire1b plants is due to a different mRNA turnover rate.
Our previous transcriptome analysis revealed that several genes are less induced in response to tunicamycin treatment in bzip60 mutant plants21. To investigate how IRE1-dependent and -independent genes are affected in bzip60, we compared the pattern of tunicamycin induction in ire1a/ire1b with that in bzip60. We first retrieved the list of tunicamycin-inducible genes in wild-type and bzip60 plants from our previous study21. After selecting 81 genes that were present in both the current and the previous gene lists (Supplementary Table S2 online), we performed a hierarchical cluster analysis using fold induction values in wild-type and ire1a/ire1b plants obtained from the current study, along with those in wild-type and bzip60 plants from the previous study21. As shown in Fig. 2a, induction of a considerable number of tunicamycin-inducible genes (63 out of 81 genes; approximately 80%) was similarly affected in both ire1a/ire1b and bzip60 mutant plants i.e. a majority of genes whose induction was reduced in ire1a/ire1b showed reduced induction in bzip60 (see Group II genes in Fig. 2a), whereas genes whose induction was unaffected in ire1a/ire1b also tended to be unaffected in bzip60 (see Group III genes in Fig. 2a). It should be noted that a certain proportion of genes showed even higher induction by tunicamycin treatment in ire1a/ire1b than in the wild-type (see Group I genes in Fig. 2a), as is evident by fold induction values in ire1a/ire1b divided by those in the wild-type being much greater than 1; no such difference was observed between the wild-type and bzip60.
The selected genes were subjected to RNA blot analysis. As shown in Fig. 2b, inductions of BiP3 and Sar1 were clearly reduced in ire1a/ire1b, whereas induction of BiP1 was still observed in ire1a/ire1b. This result was reminiscent of the pattern of transcriptional induction observed in bzip6021. A reduced induction of these genes was not observed in the ire1a or ire1b single mutant (Fig. 2c, Supplementary Fig.S3 online). This result was consistent with the observation that the ire1a/ire1b double mutant was much more sensitive to tunicamycin than the wild-type or the single mutants.
The active form of bZIP60 protein was absent in ire1a/ire1b
Our observation that IRE1A/IRE1B and bZIP60 share a similar set of transcriptional targets suggested that IRE1A/IRE1B and bZIP60 are involved in the same signalling pathway. As an initial approach to clarify their relationship, we monitored the expression profile of bZIP60 in ire1a/ire1b. As shown in Fig. 3a, transcripts of bZIP60 accumulated by tunicamycin treatment in both the wild-type and ire1a/ire1b although induction was slightly weaker in ire1a/ire1b, which is consistent with the hybridisation signals obtained from the microarray analysis (see Supplementary Table S2 online).
Subsequently, we performed immunoblot analysis using anti-bZIP60 antibody to examine the level of the active form of bZIP60 protein in ire1a/ire1b. The active, nuclear-localised form of bZIP60 protein migrating faster on an SDS-PAGE gel21 was detected by tunicamycin treatment in the wild-type but not in ire1a/ire1b (Fig. 3b). The active form of bZIP60 was detected in both ire1a and ire1b single mutants (Fig. 3c), suggesting that both IRE1A and IRE1B are involved in the production of the active form of bZIP60 protein. In addition to tunicamycin, DTT treatment also induced the active form of bZIP60 protein in the wild-type but not in ire1a/ire1b (Fig. 3d). The active form of bZIP60 protein that appeared in response to tunicamycin and DTT treatments was slightly larger than bZIP60ΔC transiently expressed in protoplasts (Fig. 3d).
The conserved stem-loop structures essential for the splicing by IRE1 were found in bZIP60 mRNA
On the basis of the aforementioned observations, we hypothesized that bZIP60 mRNA is regulated by unconventional splicing catalysed by IRE1A and IRE1B in a manner similar to the regulation of HAC1 and XBP1 mRNAs. Therefore we predicted the bZIP60 mRNA secondary structure using the CentroidFold software (http://www.ncrna.org/)24. As shown in Fig. 4a and Supplementary Fig. S4 online, bZIP60 mRNA probably forms 2 stem-loop structures observed in HAC1 and XBP1 mRNAs10,11. The conserved nucleotides in both of the 7-nucleotide loops essential for HAC1 and XBP1 mRNA splicing10,11 were also observed in bZIP60 mRNA, except that the second loop consisted of 8 instead of 7 nucleotides (Fig. 4a). Based on the splicing rule conserved in HAC1 and XBP1, we predicted that the 23-nucleotide intron is spliced out from bZIP60 mRNA during the ER stress response (Fig. 4b). We designated the unspliced and spliced mRNAs as bZIP60u and bZIP60s, respectively.
bZIP60s was predicted to produce a smaller protein (Fig. 4c, d and Supplementary Fig. S5 online), i.e. bZIP60u encodes a protein of 295 amino acids, whereas bZIP60s encodes a protein of 258 amino acids. The frameshift due to the 23-nucleotide splicing generates a new, different amino acid sequence (ORF2) in the C-terminal region of the bZIP60s protein. By converting from bZIP60u to bZIP60s, the bZIP60 protein loses the TMD that has been considered to anchor bZIP60u to the ER membrane. This estimation is consistent with the electrophoretic mobility of proteins observed in Fig. 3d in comparison to that of bZIP60ΔC, which is an artificially truncated form encoding a protein of 216 amino acids19.
BLAST searches detected sequences homologous to bZIP60 in 16 plant species. Alignment of their nucleotide sequences around the possible splicing site showed that the 6 nucleotides considered to be important for splicing were completely conserved, whereas amino acid sequences were not necessarily conserved (Supplementary Fig. S6 online). A putative intron is 23 nucleotides long in dicots, whereas it is 20 nucleotides long in monocots such as rice. A few EST sequences corresponding to a spliced form were found in currently available databases. We were unable to find any sequence corresponding to bZIP60s in Arabidopsis EST databases.
bZIP60s was detected by tunicamycin treatment in the wild-type but not in ire1a/ire1b
To test the above hypothesis, RNA was prepared from wild-type and ire1a/ire1b plants treated with tunicamycin and subjected to RT-PCR amplification using primers designed to detect an amplicon including the predicted intron (Fig. 5a). A smaller band of predicted size was detected by tunicamycin treatment in the wild-type but not in ire1a/ire1b (Fig. 5b). This band was cloned into a plasmid vector and subjected to sequencing. The determined sequence was the same as that predicted in Fig. 4.
Subsequently, primers were designed to anneal specifically to bZIP60s (Fig. 5a) and used for RT-PCR amplification. As shown in Fig. 5c, the amplicon of the predicted size was detected by tunicamycin treatment in the wild-type, but again not in ire1a/ire1b. This amplification did not appear to be affected in either of the ire1a or ire1b single mutant (Fig. 5d), indicating that both IRE1A and IRE1B are involved in the splicing of bZIP60 mRNA.
In the current study we provide evidence that Arabidopsis IRE1 homologues are involved in the ER stress response through unconventional splicing of bZIP60 mRNA. We first showed that the ire1a/ire1b double mutant, but not the ire1a or ire1b single mutant, is much more sensitive to tunicamycin than the wild-type. Microarray analysis demonstrated that induction of several tunicamycin-inducible genes is less pronounced in ire1a/ire1b than in the wild-type. Strikingly, a majority of those genes were also less induced in the bzip60 mutant. We further showed that the ire1a/ire1b double mutant does not accumulate the active form of the bZIP60 protein even when treated with tunicamycin. We noticed that bZIP60 mRNA has a secondary structure that is possibly subject to splicing by IRE1. This structure is strictly conserved in at least 16 bZIP60 homologues in plants. In particular, 6 nucleotides that have been shown to be essential in yeast HAC1 and mammalian XBP110,11 are completely conserved even though the corresponding amino acid sequences are not necessarily conserved, indicating the importance of this RNA secondary structure. We revealed that splicing of bZIP60 mRNA actually occurs in response to tunicamycin treatment. In addition, we demonstrated that bZIP60 mRNA splicing and the subsequent production of the active form of bZIP60 protein are dependent on both IRE1A and IRE1B.
We had previously concluded that bZIP60 is activated by proteolysis near the TMD in a manner similar to mammalian ATF6 and plant bZIP2821. This model arose from the fact that bZIP60 has a putative TMD (amino acids 224–244) after the bZIP domain (amino acids 140–197) and the truncated form bZIP60ΔC (amino acids 1–216) activated promoters of ER stress-inducible genes19. Our model was also supported by the observation that bZIP60u, which we previously called the full-length form, was detected in the ER fraction whereas bZIP60s, which we previously called the cleaved form, was detected in the nuclear fraction21. Importantly, however, in this study we showed that the apparent molecular mass of bZIP60s is considerably larger than that of bZIP60ΔC (Fig. 4d). Our current model nicely explains this size difference, because bZIP60u, bZIP60s and bZIP60ΔC encode proteins of 295, 258 and 216 amino acids, respectively.
Our current model is also supported by the existence of cDNA sequences of bZIP60 homologues in other plant species that lack the predicted intron (Supplementary Fig. S6 online). The boundary sequences of the intron are highly conserved among bZIP60 homologues. Most strikingly, the 3 nucleotides in both the first and second loops of 7 nucleotides essential for HAC1 and XBP1 mRNA splicing10,11 are strictly conserved, except that the second loop is of 8 rather than 7 nucleotides in plant bZIP60 homologues, thus indicating that the RNA secondary structure with the conserved ribonucleotides is essential for IRE1-dependent splicing. It is worth mentioning that RT-PCR analysis using primers amplifying both bZIP60u and bZIP60s detected much less bZIP60s than bZIP60u (Fig. 5b). This could be attributed to a stable secondary structure that bZIP60s might form, making it resistant to transcription by a reverse transcriptase. Alternatively, the spliced mRNA could actually be a minor entity in vivo.
Although some regulation of a transcription factor through unconventional splicing by IRE1 appears to also be conserved in plants, a clear difference exists in terms of the underlying molecular basis when compared to yeast and mammalian counterparts (see Fig. 6). First, although both mammals and Arabidopsis have 2 IRE1 paralogues, Arabidopsis IRE1A and IRE1B are functionally redundant in terms of bZIP60 mRNA splicing, whereas mammalian IRE1α and IRE1β have distinct functions, i.e. the former catalyses the unconventional splicing and the latter catalyses 28S ribosomal RNA cleavage10,25. Most notably, in marked contrast to yeast and mammals where HAC1s and XBP1s gain a transcriptional activation domain at their C-terminus after splicing9,10, bZIP60u already contains a transcriptional activation domain at its N-terminus20. In this regard, it is interesting to note that the XBP1u protein acts as a negative regulator26. It has been reported that a nuclear export signal and a signal for protein degradation are present only in the XBP1u protein at the C-terminal region and that the XBP1u protein forms a heterodimer with XBP1s and exports it to the cytoplasm for proteasome-dependent degradation. This is believed to downregulate the amount of XBP1s protein that needs to be degraded in the absence of ER stress. However, the same may not hold true for bZIP60u because it needs to be excluded from the nucleus in the absence of ER stress because of the presence of a transcriptional activation domain in its N-terminal region. This exclusion of bZIP60u from the nucleus is probably attributed to the TMD that anchors bZIP60u to the ER membrane, as experimentally verified in our previous study21.
Recently, Yanagitani et al. reported that the XBP1u protein tends to localise to the ER membrane through a hydrophobic region (HR) in its C-terminal region27. This anchoring of the XBP1u protein recruits XBP1u mRNA to the ER membrane where IRE1α localises and is therefore considered to achieve efficient splicing of XBP1u. A similar mechanism could be considered for bZIP60 because the TMD of bZIP60 is located immediately after the 23-nucleotide intron and the TMD gets deleted by the frameshift caused by splicing (Fig. 4c). Therefore, the putative TMD of bZIP60 might be an HR binding to the periphery of the membrane rather than a peptide region spanning the lipid bilayer. If the scenario for XBP1 is applied to bZIP60, the C-terminal region of bZIP60u may have an amino acid sequence responsible for the attenuation of translation that allows sufficient time for IRE1 to catalyse splicing28. A moderately conserved amino acid sequence in the C-terminal region of bZIP60u in several plant species might be of functional relevance for bZIP60 mRNA splicing.
It is worth mentioning that the transcriptome response of tunicamycin-inducible genes in ire1a/ire1b is not exactly the same as that in bzip60, i.e. although a majority of tunicamycin-inducible genes whose induction is reduced in ire1a/ire1b are also reduced in bzip60, a certain proportion of genes show even higher induction in ire1a/ire1b than in the wild-type whereas no such genes are observed between the wild-type and bzip60 (see Group I genes in Fig. 2a). This implies that IRE1 is not solely dedicated to splicing-mediated bZIP60 activation, but also affects other molecules. Indeed, besides an unconventional splicing of mRNA encoding a transcription factor, mammalian IRE1 has been shown to mediate mRNA decay, 28S ribosomal RNA cleavage and activation of ASK1, a mediator of ER stress-induced apoptosis25,29,30,31.
During the preparation of this manuscript, Deng et al. reported the splicing of bZIP60 mRNA32. Their RT-PCR analysis using ire1 single mutants suggests that only IRE1B accounts for splicing. However, given the differences in the tunicamycin sensitivity and in the levels of bZIP60 mRNA splicing and bZIP60s protein among the ire1 single and double mutants observed in the current study, it is evident that both IRE1A and IRE1B catalyse the unconventional splicing of bZIP60 mRNA in response to ER stress and cause a frameshift, leading to the production of the active bZIP60 transcription factor.
T-DNA insertion mutants of Arabidopsis thaliana IRE1A (SALK_018112) and IRE1B (GABI_638B07) were obtained from ABRC and GABI-Kat, respectively33,34. A T-DNA insertion mutant of bZIP60 (SALK_050203) was described previously21. These mutants and the wild-type used in this study are all in Col-0 background. Two insertion mutants (ire1a and ire1b) were crossed and the double mutant (ire1a/ire1b) was isolated from their progenies. Insertion of a T-DNA and its homozygosity were confirmed by PCR using genomic DNA and by RT-PCR as shown in Fig. 1b and Supplementary Fig. S1 online.
Nomenclature of IRE1 genes and their T-DNA insertion mutants
There are 2 independently published reports that refer to 2 Arabidopsis IRE1 homologues (AGI codes At2g17520 and At5g24360) using different names; At2g17520 and At5g24360 are referred to as IRE1-2 and IRE1-1, respectively, in one report15, whereas in the other report these are referred to as IRE1A and IRE1B, respectively14. We adopted the latter naming according to widely accepted nomenclature that is followed by the Arabidopsis research community, whose details can be found in the TAIR website (http://www.arabidopsis.org/).
With regard to the ire1 mutants, Lu and Christopher reported 3 mutants for IRE1A (ire1a-1, ire1a-2 and ire1a-3) and one mutant for IRE1B (ire1b-2)35. ire1a-2 is a T-DNA insertion mutant generated at the SALK Institute (line number; SALK_018112) and the same line was used in this study, although we independently obtained the seeds and isolated the homozygous mutant. IRE1B has been reported to be essential for embryo development as inferred from the observation that ire1b-2 homozygous mutants could not be obtained35. We were also unable to isolate ire1b-2 homozygotes. However, it is important to note that introducing a genomic DNA fragment containing the IRE1B gene did not complement the lethality of the disruption of the IRE1B gene (i.e. ire1b-2 homozygotes could not be obtained even after the IRE1B gene was introduced). Meanwhile, we obtained another T-DNA insertion mutant from GABI-Kat and were able to isolate the homozygous mutant, which we designated as ire1b-1. In the current study, we used ire1a-2 and ire1b-1 for detailed analyses, but for simplicity in the main text we designated them as ire1a and ire1b, respectively and the double mutant as ire1a/ire1b.
To examine tunicamycin sensitivity, approximately 100 seeds of each genotype were sown on MS plates containing various concentrations of tunicamycin. Two weeks later, the percentage of germinating seeds was calculated. We considered a seedling with opened cotyledons as a germinated seed. Calculations were performed using data from 3 independent experiments.
For RNA and protein extractions, sterilized seeds were sown in half-strength MS medium with 1% sucrose and allowed to grow for 10 days in a 16 h light and 8 h dark cycle at 22°C with gentle shaking. Seedlings were treated with dimethylsulfoxiside (DMSO; 0.1%) as mock treatment, tunicamycin (5 mg/l), or dithiothreitol (DTT; 2 mM) for indicated time periods and subjected to RNA and protein preparation. Although this concentration of tunicamycin eventually kills seedlings, in the time range we used in the current study, we could observe a clear transcriptomic change characteristic of the ER stress response, which is indicative of an active cellular response.
The GeneChip Arabidopsis ATH1 Genome Array (Affymetrix) representing approximately 24,000 genes was used. Seedlings of the wild-type and ire1a/ire1b grown for 10 days were treated with either tunicamycin (5 mg/l) or DMSO (0.1%) as a solvent control for 5 h and subjected to RNA preparation. RNA was extracted using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. The experiment was performed with three biological replicates. GeneChip analyses were conducted as previously described36 with the following modifications: cRNA labelling and hybridisation were conducted using the 3′INV Express kit along with the Hybridisation, Wash and Stain kit (Affymetrix) as per the manufacturer's instructions. The signal values and detection P-values were calculated using GeneChip Operating Software (Affymetrix). The data were then transformed to log scale and then statistical analyses were conducted using R as described previously36. A false discovery rate, or FDR, was analysed as described by Storey and Tibshirani37. Microarray data can be found in the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under the accession number E-MEXP-3186 “ire1 double mutant vs wild-type in response to tunicamycin”.
For the hierarchical cluster analysis, the list of tunicamycin-inducible genes in the wild-type and bzip60 plants was retrieved from our previous publication21. Among 81 genes that are present both in the current and previous gene lists (Supplementary Table S2 online), fold induction values for wild-type and ire1a/ire1b plants obtained from the current study, as well as those in wild-type and bzip60 plants from our previous study21, were calculated. The hierarchical cluster analysis was performed using MeV (http://www.tm4.org/mev/)38,39. Furthermore, a fold induction value obtained from either ire1a/ire1b or bzip60 plants was divided by that obtained from the corresponding wild-type plants and plotted in a graph format on a logarithmic scale.
RNA was extracted by using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Quantitative RT-PCR was performed as previously described40 using primers listed in Supplementary Table S3 online.
For RNA blot analysis, RNA was extracted according to the method described by Chomczynski et al.41. RNA blot was conducted by using the PCR DIG Probe Synthesis Kit (Roche) according to the manufacturer's instructions. Probes were prepared by PCR using the primers listed in Supplementary Table S3 online. RNA (5 μg per lane) was fractionated on a 1.5% agarose gel containing 2% formaldehyde, capillary blotted onto a nylon membrane (Biodyne PLUS, Pall Corporation) in 20× standard saline citrate (1×SSC = 0.15 M sodium chloride/0.015 M sodium citrate, pH7) and fixed by UV irradiation. The membrane was washed twice each with 1×SSC/0.1% SDS and 0.1×SSC/0.2% SDS at 68°C and then exposed to x-
RT-PCR to detect bZIP60s
RNA was extracted by using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. Reverse transcription was conducted at 70 °C using ThermoScript (Invitrogen). PCR was then performed at an annealing temperature of 63°C using primers bzip60iF and bzip60iR, at 68°C using primers bzip60sF and bzip60sR and at 64°C using primers bzip60sF2 and bzip60sR2.
Transient expression of bZIP60ΔC
A plasmid harbouring bZIP60ΔC under the CaMV 35S promoter was transfected in protoplasts prepared from mature leaves of the bzip60 mutant plants according to Iwata et al42.
Prediction of RNA secondary structure and protein domains
The secondary structure of bZIP60u mRNA was predicted using CentroidFold (http://www.ncrna.org/centroidfold/)24. The bZIP domain and TMD of bZIP60 were predicted according to PROSITE (http://expasy.org/prosite/)43 and TMpred (http://www.ch.embnet.org/software/TMPRED_form.html), respectively.
Alignment of bZIP60 homologues
The cDNA sequences of the Arabidopsis bZIP60u or bZIP60s were queried against the nucleotide collection and expressed sequence tag databases at the National Center for Biotechnology Information using BLASTn (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple alignments of the nucleotide sequences of unspliced and spliced forms of bZIP60 homologues were generated using ClustalW implemented in MEGA444.
Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nat Rev Mol Cell Biol 8, 519–529 (2007).
Vitale, A. & Boston, R. S. Endoplasmic reticulum quality control and the unfolded protein response: insights from plants. Traffic 9, 1581–1588 (2008).
Shamu, C. E. & Walter, P. Oligomerization and phosphorylation of the Ire1p kinase during intracellular signaling from the endoplasmic reticulum to the nucleus. EMBO J 15, 3028–3039 (1996).
Kimata, Y., Oikawa, D., Shimizu, Y., Ishiwata-Kimata, Y. & Kohno, K. A role for BiP as an adjustor for the endoplasmic reticulum stress-sensing protein Ire1. J Cell Biol 167, 445–456 (2004).
Kimata, Y. et al. Two regulatory steps of ER-stress sensor Ire1 involving its cluster formation and interaction with unfolded proteins. J Cell Biol 179, 75–86 (2007).
Mori, K., Kawahara, T., Yoshida, H., Yanagi, H. & Yura, T. Signalling from endoplasmic reticulum to nucleus: transcription factor with a basic-leucine zipper motif is required for the unfolded protein-response pathway. Genes Cells 1, 803–817 (1996).
Cox, J. S. & Walter, P. A novel mechanism for regulating activity of a transcription factor that controls the unfolded protein response. Cell 87, 391–404 (1996).
Sidrauski, C. & Walter, P. The transmembrane kinase Ire1p is a site-specific endonuclease that initiates mRNA splicing in the unfolded protein response. Cell 90, 1031–1039 (1997).
Mori, K., Ogawa, N., Kawahara, T., Yanagi, H. & Yura, T. mRNA splicing-mediated C-terminal replacement of transcription factor Hac1p is required for efficient activation of the unfolded protein response. Proc Natl Acad Sci USA 97, 4660–4665 (2000).
Yoshida, H., Matsui, T., Yamamoto, A., Okada, T. & Mori, K. XBP1 mRNA is induced by ATF6 and spliced by IRE1 in response to ER stress to produce a highly active transcription factor. Cell 107, 881–891 (2001).
Kawahara, T., Yanagi, H., Yura, T. & Mori, K. Unconventional splicing of HAC1/ERN4 mRNA required for the unfolded protein response. Sequence-specific and non-sequential cleavage of the splice sites. J Biol Chem 273, 1802–1807 (1998).
Ye, J. et al. ER stress induces cleavage of membrane-bound ATF6 by the same proteases that process SREBPs. Mol Cell 6, 1355–1364 (2000).
Haze, K., Yoshida, H., Yanagi, H., Yura, T. & Mori, K. Mammalian transcription factor ATF6 is synthesized as a transmembrane protein and activated by proteolysis in response to endoplasmic reticulum stress. Mol Biol Cell 10, 3787–3799 (1999).
Noh, S. J., Kwon, C. S. & Chung, W. I. Characterization of two homologs of Ire1p, a kinase/endoribonuclease in yeast, in Arabidopsis thaliana. Biochim Biophys Acta 1575, 130–134 (2002).
Koizumi, N. et al. Molecular characterization of two Arabidopsis Ire1 homologs, endoplasmic reticulum-located transmembrane protein kinases. Plant Physiol 127, 949–962 (2001).
Liu, J. X., Srivastava, R., Che, P. & Howell, S. H. An endoplasmic reticulum stress response in Arabidopsis is mediated by proteolytic processing and nuclear relocation of a membrane-associated transcription factor, bZIP28. Plant Cell 19, 4111–4119 (2007).
Tajima, H., Iwata, Y., Iwano, M., Takayama, S. & Koizumi, N. Identification of an Arabidopsis transmembrane bZIP transcription factor involved in the endoplasmic reticulum stress response. Biochem Biophys Res Commun 374, 242–247 (2008).
Che, P. et al. Signaling from the endoplasmic reticulum activates brassinosteroid signaling and promotes acclimation to stress in Arabidopsis. Sci Signal 3, ra69 (2010).
Iwata, Y. & Koizumi, N. An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants. Proc Natl Acad Sci USA 102, 5280–5285 (2005).
Iwata, Y., Yoneda, M., Yanagawa, Y. & Koizumi, N. Characteristics of the nuclear form of the Arabidopsis transcription factor AtbZIP60 during the endoplasmic reticulum stress response. Biosci Biotechnol Biochem 73, 865–869 (2009).
Iwata, Y., Fedoroff, N. V. & Koizumi, N. Arabidopsis bZIP60 is a proteolysis-activated transcription factor involved in the endoplasmic reticulum stress response. Plant Cell 20, 3107–3121 (2008).
Iwata, Y., Fedoroff, N. V. & Koizumi, N. The Arabidopsis membrane-bound transcription factor AtbZIP60 is a novel plant-specific endoplasmic reticulum stress transducer. Plant Signal Behav 4, 514–516 (2009).
Koizumi, N., Ujino, T., Sano, H. & Chrispeels, M. J. Overexpression of a gene that encodes the first enzyme in the biosynthesis of asparagine-linked glycans makes plants resistant to tunicamycin and obviates the tunicamycin-induced unfolded protein response. Plant Physiol 121, 353–361 (1999).
Hamada, M., Kiryu, H., Sato, K., Mituyama, T. & Asai, K. Prediction of RNA secondary structure using generalized centroid estimators. Bioinformatics 25, 465–473 (2009).
Iwawaki, T. et al. Translational control by the ER transmembrane kinase/ribonuclease IRE1 under ER stress. Nat Cell Biol 3, 158–164 (2001).
Yoshida, H., Oku, M., Suzuki, M. & Mori, K. pXBP1(U) encoded in XBP1 pre-mRNA negatively regulates unfolded protein response activator pXBP1(S) in mammalian ER stress response. J Cell Biol 172, 565–575 (2006).
Yanagitani, K. et al. Cotranslational targeting of XBP1 protein to the membrane promotes cytoplasmic splicing of its own mRNA. Mol Cell 34, 191–200 (2009).
Yanagitani, K., Kimata, Y., Kadokura, H. & Kohno, K. Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA. Science 331, 586–589 (2011).
Hollien, J. & Weissman, J. S. Decay of endoplasmic reticulum-localized mRNAs during the unfolded protein response. Science 313, 104–107 (2006).
Hollien, J. et al. Regulated Ire1-dependent decay of messenger RNAs in mammalian cells. The Journal of cell biology 186, 323–331 (2009).
Nishitoh, H. et al. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes Dev 16, 1345–1355 (2002).
Deng, Y. et al. Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis. Proc Natl Acad Sci USA 108, 7247–7252 (2011).
Alonso, J. M. et al. Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301, 653–657 (2003).
Rosso, M. G. et al. An Arabidopsis thaliana T-DNA mutagenized population (GABI-Kat) for flanking sequence tag-based reverse genetics. Plant Mol Biol 53, 247–259 (2003).
Lu, D. P. & Christopher, D. A. Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana. Mol Genet Genomics 280, 199–210 (2008).
Goda, H. et al. The AtGenExpress hormone and chemical treatment data set: experimental design, data evaluation, model data analysis and data access. Plant J 55, 526–542 (2008).
Storey, J. D. & Tibshirani, R. Statistical significance for genomewide studies. Proc Natl Acad Sci USA 100, 9440–9445 (2003).
Saeed, A. I. et al. TM4 microarray software suite. Methods Enzymol 411, 134–193 (2006).
Saeed, A. I. et al. TM4: a free, open-source system for microarray data management and analysis. BioTechniques 34, 374–378 (2003).
Iwata, Y., Sakiyama, M., Lee, M. H. & Koizumi, N. Transcriptomic response of Arabidopsis thaliana to tunicamycin-induced endoplasmic reticulum stress. Plant Biotechnol 27, 161–171 (2010).
Chomczynski, P. & Sacchi, N. The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat Prot 1, 581–585 (2006).
Iwata, Y., Lee, M. H. & Koizumi, N. Analysis of a transcription factor using transient assay in Arabidopsis protoplasts,. in Plant Transcription Factors, Vol. 754 . (eds. L. Yuan & S. E. Perry) (Humana Press, press 01).
Sigrist, C. J. et al. PROSITE, a protein domain database for functional characterization and annotation. Nucleic Acids Res 38, D161–166 (2010).
Tamura, K., Dudley, J., Nei, M. & Kumar, S. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596–1599 (2007).
We thank ABRC and GABI-Kat for providing T-DNA insertion lines. We also thank TAIR for the gene annotation data used in the present study. We thank Chitose Takahashi, Sanae Tashiro, Akiko Sato and Sachiko Ooyama for technical assistance in GeneChip analysis and Naoko Miya and Masayo Sakiyama for technical assistance in isolation of ire1 mutants. This work was supported by Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-in-Aid for Scientific Research 20380188 (to N.K.).
The authors declare no competing financial interests.
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Nagashima, Y., Mishiba, Ki., Suzuki, E. et al. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor. Sci Rep 1, 29 (2011). https://doi.org/10.1038/srep00029
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