Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

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

Several observations indicate that the RIDD pathway, if not the specific targets, is conserved in mammalian cells and Drosophila. First, the down-regulation of mouse target mRNAs was independent of XBP-1 (Fig. 1 D) . Using RNAi to deplete Ire1 from cells lacking a functional XBP-1 , we found that Ire1-dependent down-regulation of several target mRNAs occurred in the absence of XBP-1. As a control, in cells lacking XBP-1, ERdj4 was induced only to the level seen in Ire1 / cells, and this induction was insensitive to Ire1 depletion ( Fig. 1 D) . Second, the down-regulation of many mouse targets was achieved through an increase in their decay rates. We inhibited transcription with actinomycin D and monitored mRNA levels over time in the absence and presence of ER stress ( Fig. 2 and Fig. S2 ). For 7 of the 11 targets tested, the decreases in mRNA abundance were dependent not on transcription but on increased rates of mRNA degradation. Third, as in Drosophila cells, the set of targets in mouse cells is highly enriched for mRNAs encoding membrane proteins. 17 of the 22 targets in Table I that were assigned localization annotations in the mouse little effect on splicing or RIDD targets. However, cells treated with both 1NM-PP1 and ER stress degraded RIDD targets to similar levels as those expressing the wild-type Ire1 (Fig. 4 , D and E). As a further control, we confirmed the XBP-1 independence of RIDD in these cells. XBP-1 is transcriptionally induced in our cells in a largely Ire1-independent manner and therefore does not occur to a significant extent in cells treated with 1NM-PP1 alone. Therefore, we depleted XBP-1 from cells expressing Ire1-I642G using RNAi, and although this blocked containing point mutations into these cells in an isogenic manner. Using this approach, we expressed an hIre1 variant containing a single point mutation (D847A) that has been previously shown to compromise the nuclease activity of hIre1 while leaving the kinase activity intact (Tirasophon et al., 2000) . For these experiments, we used C-terminally Flag-tagged versions of both the wild-type and mutant Ire1 and confirmed by Western blotting that the hIre-D847A variant was expressed at similar levels to the wild type (Fig. 3 A) . Consistent with a lack of nuclease activity, cells expressing hIre1-D847A did not support XBP-1 splicing in the absence or presence of ER stress (Fig. 3 B) and induced ERdj4 only to the level seen in Ire1 / cells (Fig. 3, C and D) . All cell lines induced BiP by similar amounts, indicating that they are experiencing a similar level of ER stress. However, unlike the Ire1 R cells, cells expressing hIre1-D847A displayed no change in the abundance of RIDD targets upon induction of ER stress (Fig. 3, C and D) . These results indicate that the nuclease activity of Ire1 is required for degradation of RIDD targets, which is consistent with a mechanism in which Ire1 directly cleaves these RNAs in response to ER stress.