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.