Figure 8.

XBP1 deficiency induces feedback activation of IRE1α, resulting in IRE1α-mediated mRNA degradation of select p450 genes. (A) 293T cells were co-transfected with CMV SPORT6-EGFP, CMV SPORT6-Cyp1a2, CMV SPORT6-Cyp2e1, and CMV SPORT6-Bloc1s1 together with empty, WT, or K599A mutant IRE1α plasmids. Cells were harvested 24 h after transfection to measure the expression levels of the transfected genes. mRNA levels were measured by qRT-PCR and normalized to the co-transfected EGFP. Independent experiments were repeated at least three times and the representative result is shown. (B) Recombinant IRE1α was incubated with RNA substrates generated by in vitro transcription using SP6 RNA polymerase. Reaction mixtures were run on denaturing agarose gels, which were then stained with ethidium bromide. Data are representative of two independent experiments. (C) In vitro cleavage assay was performed using liver RNA as substrate. Northern blot was performed to reveal mRNA cleavage. β-Actin and Cyp2c29 mRNAs are shown as a control. Asterisks represent IRE1α-cleaved mRNA species. (D) Nucleotide sequences around IRE1α cleavage sites in Cyp1a2 and Cyp2e1 mRNAs. IRE1α cleaves G/C junctions shown in red. Matching brackets represent base pairs of the consensus secondary structure. (E) In vitro IRE1α cleavage assay of WT and mutant Cyp1a2 mRNA. (F) Predicted secondary structure of IRE1α cleavage site in Cyp1a2 mRNA. Arrowhead indicates the cleavage site. Two G residues (italicized) were changed to T to generate the mutant construct used in E. (G) In vitro IRE1α cleavage assay of WT and mutant Cyp2e1 mRNAs. Data are representative of two independent experiments.