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, 16 (4), 452-66

IRE1-mediated Unconventional mRNA Splicing and S2P-mediated ATF6 Cleavage Merge to Regulate XBP1 in Signaling the Unfolded Protein Response

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IRE1-mediated Unconventional mRNA Splicing and S2P-mediated ATF6 Cleavage Merge to Regulate XBP1 in Signaling the Unfolded Protein Response

Kyungho Lee et al. Genes Dev.

Abstract

All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed the unfolded protein response (UPR). In yeast, a type-I ER transmembrane protein kinase, Ire1p, is the proximal sensor of unfolded proteins in the ER lumen that initiates an unconventional splicing reaction on HAC1 mRNA. Hac1p is a transcription factor required for induction of UPR genes. In higher eukaryotic cells, the UPR also induces site-2 protease (S2P)-mediated cleavage of ER-localized ATF6 to generate an N-terminal fragment that activates transcription of UPR genes. To elucidate the requirements for IRE1alpha and ATF6 for signaling the mammalian UPR, we identified a UPR reporter gene that was defective for induction in IRE1alpha-null mouse embryonic fibroblasts and S2P-deficient Chinese hamster ovary (CHO) cells. We show that the endoribonuclease activity of IRE1alpha is required to splice XBP1 (X-box binding protein) mRNA to generate a new C terminus, thereby converting it into a potent UPR transcriptional activator. IRE1alpha was not required for ATF6 cleavage, nuclear translocation, or transcriptional activation. However, ATF6 cleavage was required for IRE1alpha-dependent induction of UPR transcription. We propose that nuclear-localized IRE1alpha and cytoplasmic-localized ATF6 signaling pathways merge through regulation of XBP1 activity to induce downstream gene expression. Whereas ATF6 increases the amount of XBP1 mRNA, IRE1alpha removes an unconventional 26-nucleotide intron that increases XBP1 transactivation potential. Both processing of ATF6 and IRE1alpha-mediated splicing of XBP1 mRNA are required for full activation of the UPR.

Figures

Figure 1
Figure 1
Generation and characterization of IRE1α-null MEFs. (A) Schematic representation of the predicted recombination of targeting vector and the mIRE1α locus. The bar indicates the position of a 0.5-kb BamHI–XhoI fragment used as a probe for Southern hybridization. (B) Southern analysis of ES recombinant clones (1A9 and 1H10) compared to the parental R1 cells. (C) Northern blot analysis of IRE1α-null MEFs. Wild-type and IRE1α-null MEFs were treated with or without 10 μg/mL tunicamycin for 6 h prior to harvesting total RNA for Northern blot analysis. The blot was probed with an [α-32P]-labeled 3.6-kb EcoRI–XbaI fragment from pED–hIRE1α cDNA. (D) Western blot analysis of wild-type and IRE1α-null MEFs. Proteins were prepared from wild-type and IRE1α-null MEFs (lanes 1,2) and from the pancreatic β-cell line HIT-T15 (lane 3). HIT-T15 was treated with 10 μg/mL tunicamycin for 6 h prior to protein harvest. The proteins were subjected to SDS-PAGE and Western blot analysis using anti-IRE1α lumenal-domain antibody. Phosphorylated and nonphosphorylated forms of IRE1α are indicated. (E,F) Northern blot analysis of wild-type and IRE1α-null MEFs. Wild-type, heterozygous, or homozygous IRE1α-null MEFs were treated with or without 10 μg/mL tunicamycin for 6 h prior to harvesting total RNA for Northern blot analysis. One blot was probed with [α-32P]-labeled hamster BiP cDNA and β-actin cDNA (E), and another blot was probed with [α-32P]-labeled mouse GRP94 DNA and β-actin cDNA (F). Quantification of the results showed that tunicamycin induced GRP94 mRNA 6.7-fold and 5.4-fold in wild-type and IRE1α-null MEFs, respectively. (G) BiP reporter gene expression in IRE1α-null MEFs. The reporter plasmids containing the luciferase gene under control of the rat BiP promoter and β-galactosidase under control of the CMV promoter were cotransfected into wild-type and IRE1α-null MEFs. The transfected cells were treated with 2 μg/mL tunicamycin for 16 h prior to harvest. The luciferase activities are presented relative to CMV β-galactosidase activities. Similar results were obtained from three independent experiments.
Figure 2
Figure 2
5× ATF6 reporter activation is defective in IRE1α-null MEFs. (A) 5× ATF6 reporter gene expression in wild-type and IRE1α-null MEFs. The reporter plasmids containing the luciferase gene under control of 5× ATF6 binding sites and β-galactosidase under control of the CMV promoter were cotransfected into wild-type and IRE1α-null MEFs. The transfected cells were treated with 2 μg/mL tunicamycin for 16 h prior to harvest. The luciferase activities are presented relative to CMV β-galactosidase activities. Similar results were obtained from two independent experiment. (BD) Wild-type and IRE1α-null MEFs were transfected as in A in the presence of vector alone or vector encoding wild-type IRE1α, kinase-defective (K599A) IRE1α, RNase defective (K907A) IRE1α, C-terminal-deleted IRE1α (IRE1αΔC), ATF2, ATF4, ATF6, processed form of ATF6 (ATF6 50-kD), c-Jun, or c-Fos as indicated. The vector used for IRE1α expression was pEDΔC. The empty vectors used as controls were pEDΔC (B,D), pcDNA3 (Vector 1), and pCMV-HA (Vector 2) (C). MEFs were transfected by either Effectine (B,D) or FuGENE6 (C) according to the manufacturers' recommended procedures. The transfected cells were treated with 10 μg/mL tunicamycin for 6 h (B,D) or 2 μg/mL tunicamycin for 16 h (C) prior to harvest. Similar results were obtained from four independent experiments.
Figure 3
Figure 3
IRE1α is not required for ATF6 cleavage, nuclear translocation, or transcriptional activation. (A) Western blot analysis of ATF6. Wild-type and IRE1α-null MEFs were treated with tunicamycin (10 μg/mL) for increasing times, and protein extracts were prepared for Western blot analysis. ATF6 proteins were detected using anti-ATF6 antibody and anti-rabbit immunoglobulin conjugated with horseradish peroxidase and enhanced chemiluminescence. (B) Pulse-chase analysis of ATF6. Wild-type and IRE1α-null MEFs were pulse-labeled with [35S]methionine and [35S]cysteine (0.5 mCi/100-mm dish) for 40 min, and then chase was performed with or without 10 μg/mL tunicamycin for the periods indicated. Proteins were extracted and immunoprecipitated using anti-ATF6 antibody. Immunoprecipitates were subjected to SDS-PAGE, and radiolabeled proteins were visualized using PhosphorImager (Molecular Dynamics). (C) ATF6 cleavage-dependent GAL4 reporter gene expression in wild-type and IRE1α-null MEFs. The reporter plasmids containing the luciferase gene under control of the GAL4 promoter and β-galactosidase under control of the CMV promoter were cotransfected with the GAL4 DNA-binding domain–ATF6 fusion protein expression vector into wild-type and IRE1α-null MEFs. The transfected cells were treated with 2 μg/mL tunicamycin for 16 h prior to harvest. The luciferase activities are presented relative to CMV β-galactosidase activities. Similar results were obtained from two independent experiments. The diagram on the left depicts ATF6 cleavage-dependent GAL4 reporter gene expression that is independent from the transcriptional activity of endogenous ATF6.
Figure 4
Figure 4
5× ATF6 reporter induction requires IRE1α-dependent splicing of XBP1 mRNA. (A) Alignment of ATF6, XBP1, CREB, and ERSE DNA sequence motifs. The entire oligonucleotide sequence used to construct the 5× ATF6 reporter is shown. The 5′ sequence located outside of the boxed region is the fixed flanking sequence used to generate random oligonucleotides (Wang et al. 2000). (B) Schematic representation of unspliced and spliced forms of the murine XBP1 mRNA and protein coding regions. The translated portion of the two open reading frames, the 26-bp intron, and the bZIP domains are depicted. (C) The predicted mRNA secondary structure at the splice-site junctions in XBP1 mRNA. The 3 residues important for cleavage of HAC1 mRNA by Ire1p (−1G, −3C, and +3G) are conserved in the 5′ and 3′ loops. (D) RT–PCR analysis of XBP1 mRNA splicing using RNA templates from tunicamycin-treated wild-type and IRE1α-null MEFs. (E) Western blot analysis of XBP1. Cell extracts were prepared from wild-type and IRE1α-null MEFs cultured in the presence or absence of tunicamycin (10 μg/mL) with MG132 (10 μM) for increasing times as indicated. (F) Northern blot analysis of XBP1 mRNA in IRE1α-null MEFs. Wild-type and IRE1α-null MEFs were treated with or without 10 μg/mL tunicamycin for 6 h prior to harvesting total RNA for Northern blot analysis. The blots were probed with the [α-32P]-labeled 0.94-kb XhoI fragment of XBP1-u and β-actin cDNA. Quantification of the results showed 3.1-fold and 4.0-fold induction with tunicamycin treatment in wild-type and IRE1α-null MEFs, respectively. (G) Western blot analysis of XBP1. The 5× ATF6 reporter plasmid and β-galactosidase under control of the CMV promoter were cotransfected into COS-1 cells in the presence of the CMV-promoter-driven unspliced form of XBP1 (XBP1-u), the spliced form of XBP1 (XBP1-s), or the first ORF of XBP1 (XBP1–ORF1) as indicated. Cells were treated with or without tunicamycin (2 μg/mL) for 8 h before harvest. Lactacystin (10 μM) was added to the media for the final 2 h. An XBP1-reactive polypeptide likely derived from using a cryptic 3′ splice site is indicated with an asterisk. (H) The 5× ATF6 reporter is activated by IRE1α-dependent XBP1 mRNA splicing. Wild-type and IRE1α-null MEFs were transfected and assayed as described in Figure 2 in the presence of the CMV-promoter-driven unspliced form of XBP1 (XBP1-u), the spliced form of XBP1 (XBP1-s), or the first ORF of XBP1 (XBP1–ORF1).
Figure 5
Figure 5
IRE1α cleaves both splice-site junctions in XBP1 RNA in vitro and is localized to the inner nuclear envelope. (A) 32P-labeled wild-type and mutant XBP1 RNAs were prepared and incubated with immunoprecipitated wild-type or RNase-defective (K907A) IRE1α protein in nuclease buffer and analyzed by electrophoresis on a denaturing polyacrylamide gel. The 5′ exon (114 nt), intron (26 nt), and 3′ exon (305 nt) cleavage products of the substrate are marked on the left. The numbers on the right are the expected nucleotide sizes. (B) Intracellular localization of IRE1α. Wild-type and IRE1α-null MEFs were fractionated as described in Materials and Methods. Western blot analysis was performed with mouse anti-IRE1α, human anti-lamin B receptor, or rabbit anti-calreticulin antibodies. (Lane 1) Cellular extract; (lane 2) nuclei with inner nuclear membrane; (lane 3) Triton X-100 soluble, microsomal, and outer nuclear membrane fraction.
Figure 6
Figure 6
IRE1α-mediated induction of UPR genes requires ATF6 cleavage. (AC) BiP reporter gene (A,C) and 5× ATF6 reporter gene (B,C) expression in S2P-deficient CHO cells. The reporter plasmids containing the luciferase gene under control of the rat BiP promoter or the 5× ATF6 binding sites were cotransfected with β-galactosidase under control of the CMV promoter and an IRE1α (A,B) or ATF6 (C) expression vector into S2P-deficient CHO cells. Immunoglobulin μ heavy chain (μ) and mutant immunoglobulin μ heavy chain deleted of the signal peptide (Δsμ) were used as positive and negative ER stress inducers, respectively. At 32 h posttransfection, cells were treated with 2 μg/mL tunicamycin for 16 h prior to harvest. The luciferase activities are presented relative to CMV β-galactosidase activities. Similar results were obtained from two independent experiments. (D) Western blot analysis of BiP. Wild-type and S2P-deficient CHO cells were transfected with plasmids as indicated. At 32 h posttransfection, the transfected cells were treated with 2 μg/mL tunicamycin for 16 h, harvested, and analyzed by Western blot analysis using anti-BiP antibody. (E) Northern blot analysis of BiP and XBP1 mRNA in S2P-deficient cells. Wild-type and S2P-deficient CHO cells were treated with or without 2 μg/mL tunicamycin for 16 h prior to harvesting total RNA for Northern blot analysis using hamster BiP, XBP1-u, and β-actin cDNAs as probes. Quantification of the results showed that tunicamycin induced BiP mRNA 34-fold and 2.6-fold and XBP1 mRNA 3.1-fold and 2.9-fold in wild-type and S2P-deficient CHO cells, respectively.
Figure 7
Figure 7
ATF6- and IRE1α-dependent UPR signaling pathways merge through regulation of the quantity and quality, respectively, of XBP1 protein. The model depicts the activation of two proximal sensors of the UPR, ATF6 and IRE1α, upon ER stress. Upon accumulation of unfolded proteins in the ER lumen, ATF6 leaves the ER to enter the Golgi apparatus, where it is cleaved by S1P and then S2P to release a 50-kD fragment that enters the nucleus through the nuclear pore. p50-ATF6 then interacts with ERSE motifs to activate transcription. Simultaneously and independently, the UPR induces dimerization, autophosphorylation, and activation of the RNase activity of IRE1α that is localized at the inner leaflet of the nuclear envelope. Activated IRE1α then initiates splicing of XBP1 mRNA to generate a potent transcriptional activator, XBP1-s, that also enters the nuclear pore to activate transcription from ERSE motifs. The status of XBP1-s and p50-ATF6 when bound to the ERSE is not known, but for simplicity they are depicted as heterodimers.

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