1. Volume 107, Issue 7, Pages 881-891 (December 2001)
XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor 
Hiderou Yoshida, Toshie Matsui, Akira Yamamoto, Tetsuya Okada, Kazutoshi Mori 
Cell 
Volume 107, Issue 7, Pages (December 2001)
DOI: /S (01)

2. Figure 1 XBP1 mRNA Is Spliced in Response to ER Stress
(A) Time course of induction of XBP1. HeLa cells were treated with 2 μg/ml tunicamycin and lysates were analyzed by immunoblotting using anti-XBP1-A (upper), anti-XBP1-B (middle), and anti-ATF6α (lower) antibodies. In vitro translation product of XBP1 mRNA as well as ATF6α (1–373) translated in vitro were run on the same gel for comparison. The migration positions of full-range rainbow molecular weight markers (Amersham Pharmacia Biotech) are indicated. The asterisk denotes a nonspecific band.
(B) Schematic presentation of ORFs encoded by XBP1 mRNA. Locations of the two ORFs (ORF1 and ORF2) in human (upper) and murine (lower) XBP1 mRNA are marked by closed boxes and the percent identities between them are shown on the right. Numbers above the respective mRNA denote nucleotide positions with the transcription start site set at 1.
(C) Splicing of XBP1 mRNA upon ER stress. Left, schematic structures of RT-PCR products from unspliced and spliced XBP1 mRNA are shown. Right, HeLa cells were treated with 300 nM thapsigargin and total RNA prepared was amplified by RT-PCR. Resulting products were subjected to 2% agarose gel electrophoresis (upper panel). The lengths of DNA size markers are shown on the left. Aliquots of total RNA were also analyzed by Northern blot hybridization (lower panel).
(D) Nucleotide sequences of cDNA corresponding to unspliced and spliced XBP1 mRNA around the splicing sites. Spliced versions of XBP1 cDNA can be found in the expressed sequence tag (EST) databases as indicated.
Cell  , DOI: ( /S (01) )

3. Figure 2 The C-Terminal Region of XBP1 Is Replaced by Splicing
(A) Schematic representation of the unspliced and spliced forms of XBP1 (pXBP1(U) and pXBP1(S), respectively). Numbers indicate amino acid positions with the initiation methionine set at 1. Numbers with asterisks imply that these aa are encoded by ORF2. Basic and leucine zipper (ZIP) domains, as well as the three regions used to raise specific antibodies are indicated.
(B) Direct evidence for C-terminal replacement of XBP1 induced by ER stress. HeLa cells were treated with 300 nM thapsigargin and lysates were analyzed by immunoblotting using anti-XBP1-A (upper), anti-XBP1-C (middle), and anti-ATF6α (lower) antibodies together with in vitro-translated pXBP1(U) and pXBP1(S). The bands with asterisks are nonspecific.
(C) RNase protection assay. Upper panel, the probe used and protected fragments expected after RNase digestion are presented schematically. Lower panel, radiolabeled RNA probe was hybridized with yeast RNA or total RNA prepared from HeLa cells that had been treated with or without 300 nM thapsigargin or 2 μg/ml tunicamycin, digested with RNase, and subjected to 5% polyacrylamide-8 M urea gel electrophoresis. The lengths of DNA size markers are shown on the left.
Cell  , DOI: ( /S (01) )

4. Figure 3 Properties of Unspliced and Spliced Forms of XBP1
(A) Stability of pXBP1(U) and pXBP1(S). HeLa cells transfected with pcDNA-XBP1(unspliced) and pcDNA-XBP1(spliced) simultaneously were pulse-labeled with 35S-methionine and 35S-cysteine, and then chased. pXBP1(U) and pXBP1(S) immunoprecipitated using anti-XBP1-A antibody were subjected to SDS-PAGE. Radioactivities of each band were determined and data are plotted on the right as relative abundance (arbitrary units) versus chase time.
(B) Effects of pretreatment with MG132 on the levels of pXBP1(U) and pXBP1(S). HeLa cells were pretreated with (+) or without (−) 10 μM MG132 for 2 hr and then treated with 300 nM thapsigargin (Tg) without removing MG132. Lysates were analyzed by immunoblotting using anti-XBP1-A antibody together with in vitro-translated pXBP1(U) and pXBP1(S).
(C) Binding of pXBP1(U) and pXBP1(S) to ERSE. 32P-labeled ERSE-CC containing CCAAT-N9-CCACG was incubated with various forms of XBP1 or ATF6α(1–373) translated in vitro in the presence (+) or absence (−) of recombinant NF-Y trimer. Protein-DNA complexes formed were separated from free DNA probe by electrophoresis on a nondenaturing gel.
(D) Supershift experiments. A mixture of in vitro-translated pXBP1(S) and recombinant NF-Y trimer was treated with (+) or without (−) various antisera prior to incubation with 32P-labeled ERSE-CC.
(E) Competition experiments. The binding of in vitro-translated pXBP1(S) and recombinant NF-Y trimer to 32P-labeled ERSE-CC was competed by a 100-fold molar excess of various unlabeled ERSE oligonucleotides. CM, MC, and MM contain CCAAT-N9-gatgt, gacta-N9-CCACG, and gacta-N9-gatgt, respectively.
Cell  , DOI: ( /S (01) )

5. Figure 4 The Spliced Form of XBP1 Has Higher Transcriptional Activity
(A) Effects of overexpression of pXBP1(U) and pXBP1(S) on the UPR. (a) Schematic structures of pXBP1(U) and pXBP1(S). (b) Reporter assay. HeLa cells were transfected with pcDNA-XBP1(unspliced), pcDNA-XBP1(spliced), or pcDNA-ATF6α(1–373) together with the reporter plasmid pGL3-GRP78P(−132)-luc. The relative luciferase activity in transfected cells incubated for 12 hr in the presence (solid boxes) or absence (open boxes) of 2 μg/ml tunicamycin was determined, and averages from four independent experiments are presented with standard deviations (error bars). (c) Levels of pXBP1(U) and pXBP1(S). Lysates of transfected HeLa cells were analyzed by immunoblotting using anti-XBP1-A antibody. (d) Levels of GRP78 and GAPDH mRNA. Total RNA was prepared from transfected HeLa cells and analyzed by Northern blot hybridization.
(B) Characterization of the transactivation domain of XBP1. (a) Schematic structures of plasmids analyzed. (b) Reporter assay. HeLa cells were transfected with each of the plasmids shown in (a) together with the reporter plasmid pG5luc. The relative luciferase activity constitutively expressed in transfected cells was determined and presented as in (A) on the left. The averages of the relative activities are also presented on the right after correction by the level of each full-length fusion protein detected in transfected cells. (c) Levels of fusion proteins. Lysates of HeLa cells transfected with each of the plasmids shown in (a) were analyzed by immunoblotting using anti-GAL4BD antibody. The bands with asterisks denote the full-length fusion protein in each case.
Cell  , DOI: ( /S (01) )

6. Figure 5 Human IRE1α Is Involved in Splicing of XBP1 mRNA
(A) Secondary structures of the splice sites of human XBP1 and yeast HAC1 mRNA. The cleavage sites in HAC1 mRNA are indicated by arrows and six nucleotides indispensable for the cleavage reaction are boxed.
(B) Effects of overexpression of IRE1α on splicing of XBP1 mRNA. HeLa cells were transfected with pcDNA-XBP1(unspliced) together with (+) or without (−) pcDNA-IRE1α. Lysates were prepared 24 hr after transfection and analyzed by immunoblotting using anti-XBP1-A antibody together with in vitro-translated pXBP1(U) and pXBP1(S). Various point mutations were introduced into the 5′ (upper panel) and 3′ (lower panel) splice sites and resulting mutant XBP1 expression plasmids were analyzed similarly.
(C) Effects of cooverexpression of IRE1α and unspliced XBP1 mRNA on the UPR. HeLa cells were transfected in combination with pcDNA (vector), pcDNA-XBP1(unspliced), or pcDNA-IRE1α together with the reporter plasmid pGL3-GRP78P(−132)-luc. The relative luciferase activities in transfected cells incubated for 12 hr in the presence (solid boxes) or absence (open boxes) of 300 nM thapsigargin were determined and presented as in Figure 4A.
Cell  , DOI: ( /S (01) )

7. Figure 6 IRE1-Dependent Signaling Is Important for the UPR
(A) Characterization of the IRE1-dependent splicing system. HeLa cells were transfected in combination with pcDNA (vector), pcDNA-XBP1(unspliced), pcDNA-IRE1α, or pcDNA-IRE1α-ΔC. Lysates of transfected cells incubated for 6 hr with (+) or without (−) 300 nM thapsigargin were analyzed by immunoblotting using anti-XBP1-A antibody together with in vitro-translated pXBP1(U).
(B) Dependence of XBP1 production on the dose of XBP1 mRNA. HeLa cells were transfected with 5 μg of pcDNA-IRE1α and increasing amounts (a series of 2-fold dilutions) of pcDNA-XBP1(unspliced) or treated for 6 hr with (+) or without (−) 300 nM thapsigargin. Lysates were analyzed by immunoblotting using anti-XBP1-A antibody.
(C) Effects of dominant-negative mutants of IRE1α and ATF6α on ERSE- and ATF6 site-mediated transcription. HeLa cells were transfected with 0.2 μg of pcDNA (vector), pcDNA-IRE1α-ΔC, or pcDNA-ATF6α(171–373) together with the reporter plasmid pGL3-GRP78P(−132)-luc (upper panel) or p5xATF6GL3 (lower panel). The relative luciferase activities in transfected cells incubated for 12 hr in the presence (solid boxes) or absence (open boxes) of 300 nM thapsigargin were determined and presented as in Figure 4A.
(D) Dose dependence of dominant-negative effects of IRE1α-ΔC and ATF6α(171–373) on ERSE- and ATF6 site-mediated transcription. HeLa cells were transfected with various amounts of pcDNA-IRE1α-ΔC (open circles) or pcDNA-ATF6α(171–373) (closed circles) together with pGL3-GRP78P(−132)-luc (upper panel) or p5xATF6GL3 (lower panel). The relative luciferase activities in transfected cells incubated for 12 hr in the presence or absence of 300 nM thapsigargin were determined. The fold induction was plotted against the dose of each plasmid transfected.
(E) Binding of ATF6 and XBP1 to the ATF6 site. 32P-labeled ERSE-CC, ATF6 site, or ATF6 site mutant was incubated with ATF6α(1–373) or pXBP1(S) translated in vitro in the presence (+) or absence (−) of recombinant NF-Y trimer. Protein-DNA complexes formed were separated as in Figure 3C. Supershift experiments were carried out as in Figure 3D.
Cell  , DOI: ( /S (01) )

8. Figure 7 Model for the Mammalian UPR
Cell  , DOI: ( /S (01) )