To dissect the effects of IRE1α’s catalytic activities on TXNIP upregulation, we tested two point mutants. The first, IRE1α (I642G), has an enlarged adenosine triphosphate (ATP)-binding pocket in its kinase domain that destroys phosphotransfer catalytic activity; the enlarged pocket can selectively bind 1NM-PP1, a cell-permeable adenosine nucleotide mimic with a bulky chemical head group (Figure 2C) (Han et al., 2008; Papa et al., 2003). Binding of 1NM-PP1 to IRE1α (I642G) allosterically activates the RNase domain, causing it to forcibly splice XBP1 mRNA, while bypassing the auto-phosphorylation requirement (Figure S4B and C). A second mutant, IRE1α (N906A), can properly autophosphorylate when expresssed, but because its RNase active site is mutated cannot splice XBP1 mRNA (Figures 2C, F and S4C). Interestingly, expression of IRE1α (N906A) leads to small, reproducible decreases in basal TXNIP mRNA (Figure 2D and Figure S4E) and protein (Figure 2F), consistent with its known dominant-negative effects against endogenous IRE1α. Furthermore, induction of either IRE1α (I642G)—or forced expression of spliced XBP1 transcription factor (XBP1s)—cause minimal elevation of TXNIP mRNA, without discernible changes in TXNIP protein (Figure 2D–G). These results argue
