Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

Hollien, Julie; Lin, Jonathan H.; Li, Han; Stevens, Nicole; Walter, Peter; Weissman, Jonathan S.

Recently, it has been shown that for yeast and human Ire1, the cytoplasmic kinase activity can be bypassed using a small molecule that binds to the enlarged ATP pocket of an engineered Ire1 variant (Papa et al., 2003; Lin et al., 2007) . This variant, I642G in hIre1, has very little activity in the absence of the ATP analogue 1NM-PP1 (4-amino-1-tert-butyl-3-[1'-naphthyl methyl]pyrazolo[3,4-d] pyrimidine). Binding allows activation of the nuclease while simultaneously inhibiting the kinase activity of Ire1-I642G. In HEK293 cells (Lin et al., 2007) and MEF cells (Han et al., 2008) , hIre1-I642G splices XBP-1 and induces the UPR upon binding 1NM-PP1 alone, even in the absence of ER stress. To test whether activation of Ire1 is sufficient for degradation of RIDD substrates, we introduced hIre1-I642G into Ire1 / cells and tested its activity in the absence and presence of 1NM-PP1 and ER stress (Fig. 4) . Treatment of mouse cells expressing hIre1-I642G with 1NM-PP1 was sufficient to induce splicing of XBP-1 (Fig. 4 A) and transcriptional induction of the Ire1/XBP-1 target ERdj4 (Fig. 4 B) . This Ire1 activity was not caused by a general induction of ER stress, as cells lacking Ire1 or expressing the wild-type hIre1 were insensitive to 1NM-PP1 (Fig. 4) , although the drug did suppress the Ire1-independent induction of ERdj4 by DTT in all strains through an unknown mechanism (Fig. 4 B) . The 1NM-PP1induced activation of hIre1-I642G was not caused by increased levels of the protein, as assayed by Western blotting (Fig. 4 C) . Thus, our data are consistent with a 1NM-PP1-induced conformational change in the Ire1 variant itself, as proposed previously (Papa et al., 2003) .