1. Volume 34, Issue 2, Pages 191-200 (April 2009)
Cotranslational Targeting of XBP1 Protein to the Membrane Promotes Cytoplasmic Splicing of Its Own mRNA 
Kota Yanagitani, Yusuke Imagawa, Takao Iwawaki, Akira Hosoda, Michiko Saito, Yukio Kimata, Kenji Kohno 
Molecular Cell 
Volume 34, Issue 2, Pages (April 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 The XBP1u mRNA Is Distributed to the Membrane
(A) Total (T), digitonin-extracted (cytosolic, Cy), and residual (membrane-bound, M) lysates (10 μg protein) of HEK293T cells were analyzed by western blotting using anti-Calnexin (CNX) or anti-GAPDH antibodies.
(B) Total RNA samples (10 μg RNA) prepared from the lysates used in A were analyzed by northern blotting.
(C) The membrane-localization values for each mRNA shown in (B) are presented as averages and standard deviations of triplicate measurements.
(D) Total RNA samples from HEK293T cells treated (+) with 1μg/ml thapsigargin (Tg) for 1 hr or left untreated (−), and then subjected to RT-PCR to amplify XBP1 fragments containing the IRE1α-mediated splicing site. The positions of the RT-PCR products representing the unspliced (u) and the spliced (s) mRNAs are indicated.
(E) Total RNA samples (10 μg RNA for northern blotting and 2.5 μg RNA for RT-PCR) prepared from digitonin-extracted (Cy) and residual (M) lysates of untreated or tunicamycin (Tm; 2 μg/ml 6 hr) or thapsigargin (Tg; 0.5 μg/ml 6 hr)-treated HEK293T cells were analyzed by northern blotting or RT-PCR using the same primer sets as (D). The positions of the RT-PCR products representing the unspliced (u) and spliced (s) mRNAs are indicated. The membrane-localization values of XBP1 mRNA described in Experimental Procedures are presented in the right panel as averages and standard deviations of triplicate measurements.
(F) Twenty-four hours after transfection of an XBP1u or an XBP1s-expression plasmid (pcDNA3.1-XBP1u or pcDNA3.1-XBP1s) or an empty vector (pcDNA3.1), HEK293T cells were subjected to RNA analysis as in (B). Minor variants of plasmid-expressed XBP1 mRNA, which were neglected in the following experiments, are marked by an asterisk. The position of endogenous XBP1 mRNA is indicated by an arrow.
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3. Figure 2
Recruitment of the XBP1u mRNA to the Membrane Depends on Its Translatable 3′ Segment
(A) Except for construction number (const. num.) 19 and 20, XBP1 constructs carrying the indicated mutations at the splicing site were expressed in HEK293T cells by transient transfection of their expression plasmids generated from pcDNA3.1. Nucleotide sequences of the XBP1u cDNA position 481 to 534 (where 1 is the A of the initiation methionine codon) and of the same regions of the mutant and XBP1s cDNAs are presented. The red and the blue letters, respectively, represent the intron sequence and mutations (nucleotide substitutions for 5′D-stem, 3′D-stem, 5′G(−1)C and 3′G(−1)C, and insertion for Int(+A)). The XBP1u mRNA is predicted to form two stem-loop structures, as marked by green lines, both of which are required for cleavage by IRE1α. Also shown are the stem-loop (SL) numberings and the numbers representing the translational reading frame of the 3′ segment (Frame; that of the XBP1u mRNA is set at 1). The data shown in Figures 1B, 1C, and 2B are summarily presented as location (Loc.) of the mRNA (M, membranous; Cy, cytosolic; nt, not tested). (B and C) The cellular localization of plasmid-expressed XBP1 mRNA variants were examined as shown in Figure 1E, and the membrane-localization values of the plasmid-expressed XBP1 mRNA variants were calculated from triplicate experiments and represented in the lower panel, respectively. (D and F) Cellular localization of the plasmid-expressed mRNA variants was checked and presented as in (B). (E) The indicated constructs were inserted into pcDNA3.1 for expression in HEK293T cells by transient transfection. Nucleotide positions are numbered based on the structure of the XBP1u mRNA, where 1 is the A of the initiation methionine codon. For reference, the structure of the XBP1u protein is also shown. The shaded boxes represent hydrophobic regions (see Figure S1A). The red squares represent the intron. The red lines depict the 3′-UTR and the purple squares represent sequences attached for RT-PCR amplification using the primer sites represented by the arrows. The blue box indicates the ER-targeting signal peptide of calreticulin. For insertion mutants, the inserted nucleotides are shown in blue letters. The numbers in the right-hand column designated “Frame” refer to the translational reading frame of the 3′ segment of the XBP1u mRNA, with the normal frame of XBP1u mRNA set at 1. The protein products of const. num. 1, 12, 14, and 16 terminate at the termination codon term(u), whereas those of const. num. 6 and 15 terminate at term(s).
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4. Figure 3
Membrane Localization of the XBP1u mRNA Contributes to its Effective Splicing by IRE1α
(A) Deduced secondary structure of the splicing site of the XBP1u mRNA. The blue letter indicates the nucleotide inserted in the Int(+A) mutation. The intron is shown by red letters.
(B and C) Twenty-four hours after transfection of pcDNA3.1-XBP1u-ps (for expression of const. num. 16), pcDNA3.1-Int(+A)-ps (for expression of const. num. 17), or pcDNA3.1-SS-Int(+A)-ps (for expression of const. num. 18), HEK293T cells were checked for cellular localization of the mRNA products. The values in (C) represent mean and standard deviation (SD) of three independent experiments.
(D) The unspliced and the indicated mutant XBP1 RNAs generated by in vitro transcription in the presence of 32P-CTP were subjected to in vitro cleavage reaction by IRE1α. The products were electrophoretically separated on a urea-containing polyacrylamide gel and visualized by autoradiography.
(E) Twenty-four hours after transfection of pcDNA3.1-XBP1u-ps, pcDNA3.1-Int(+A)-ps, or pcDNA3.1-SS-Int(+A)-ps, HEK293T cells were treated with 0.5 μg/ml Tg for 4 hr (+) or left untreated (−). Their total RNAs were analyzed by RT-PCR using the primer set shown in Figure 2E (arrows).
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5. Figure 4
XBP1u mRNA Is Recruited to the Membrane as a Nascent XBP1u Polypeptide-Ribosome-mRNA Complex
(A and B) Twenty-four hours after transfection of pcDNA3.1-XBP1s-ORF (for expression of const. num. 15; lanes 1 and 2) or pcDNA3.1-XBP1u-ORF (for expression of const. num. 12; lanes 3 and 4) or cotransfection of equal amounts of these two plasmids (lanes 5 and 6), HEK293T cells were examined for cytosolic (Cy) or membrane (M) localization of their respective mRNA products. Membrane localization values were calculated from the RNAs detected in (A) and presented in (B).
(C and D) Twenty-four hours after transfection of pcDNA3.1-XBP1u, HEK293T cells were treated with 1 mM puromycin (Puro) or 1 mM cycloheximide (CHX) for 15 min, and checked for cellular localization of the XBP1 mRNA product. The values in (D) represent mean and SD of three independent experiments.
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6. Figure 5 Membrane Association of the XBP1u Protein
(A) Twenty-four hours after transfection of plasmids expressing FLAG-tagged XBP1s or XBP1u (pcDNA3.1-F-XBP1s or pcDNA3.1-F-XBP1u), HEK293 cells were separated into nuclear (N) and postnuclear (cytoplasmic; Cp) fractions, and equal amounts of protein lysates were analyzed by western blotting to detect the indicated epitope or proteins. (u) and (s) represent the positions of unspliced and spliced form of XBP1 protein, respectively.
(B) The cells in (A) were separated into mitochondrial (Mt), microsomal (Ms), and cytosolic (Cy) fractions, and analyzed by western blotting to detect the indicated epitope or proteins. The asterisk indicates the position of a nonspecifically detected protein.
(C) The postmitochondrial fraction (containing microsomal and cytosolic components) obtained from experiment B was incubated with the final concentration of the indicated reagents for 1 hr at 4°C on ice, separated into pellet (P) and supernatant (S) fractions by ultracentrifugation, and analyzed by western blotting to detect the indicated epitope or proteins. The asterisk indicates the position of a nonspecifically detected protein.
(D and E) 35S-labeled XBP1u or XBP1s proteins were produced in an in vitro translation system using rabbit reticulocyte lysate in the presence or absence of CMM. The reaction mixtures were separated by ultracentrifugation, and pellet (P) and soluble (S) fractions were analyzed by SDS-PAGE followed by autoradiography. In (E), the indicated amounts of CMM were added to the 25 μl reaction mixture, and radioactive signal intensity was quantified and presented in the right-hand panel.
(F) In vitro translated mixture (+ CMM), generated as in (D), was incubated with the indicated concentrations of NaCl at 4°C for 1 hr, and separated into pellet and soluble fractions.
(G) Experiment similar to that shown in (D), except that liposomes (Lipo) were used instead of CMM.
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7. Figure 6
Involvement of a Hydrophobic Region of the XBP1u Protein in the Membrane Localization of Its mRNA
(A) The indicated constructs were inserted into pcDNA3.1 and expressed in HEK293T cells by transient transfection for the experiments in (C), (E), and (G) to examine mRNA distribution, or their open reading frames were inserted into pBS2-SK(−) for in vitro transcription translation for the experiments in (B), (D), and (F) to examine protein distribution. For reference, the structure of the XBP1u protein is also shown. The shaded boxes represent hydrophobic regions. See the legend to Figure 1E for other features.
(B–G) The XBP1u multiple point mutations HR2-mut1 and HR2-mut2 (see Figure S1B for positions) and the mutations shown in (A) were tested for their effect on the ability of the XBP1u proteins to associate with CMM in vitro, as shown in Figures 5D and 5E (B, D, and F), or to examine the cellular localization of the XBP1u mRNA (C, E, and G). The values represent mean and SD of three independent experiments.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7
IRE1α Is Not Required for Membrane Localization of the XBP1u mRNA
(A) Wild-type and IRE1α KO MEF cells were treated with 0.5 μg/ml Tg for 4 hr (+) or left untreated (−), and their total RNA samples were analyzed by northern blotting and RT-PCR using the same primer set as shown in Figure 3D. The positions of the RT-PCR products from the unspliced (u) and spliced (s) XBP1 mRNAs are indicated.
(B) The cells were subjected to ER stress, or not stressed, as in (A), and analyzed for mRNA localization.
(C) Our model depicting recruitment of the XBP1u mRNA to the membrane, where it is spliced by IRE1α. Nascent XBP1u polypeptide associates with lipid membrane via the HR2 sequence. This in turn tethers XBP1u mRNA to the membrane, where it can be catalyzed by IRE1α.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions