Regulated Ire1-dependent decay of messenger RNAs in mammalian cells

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

To determine whether the lack of RIDD in our 1NM-PP1induced cells is merely a threshold effect, we treated cells with higher concentrations of 1NM-PP1, such that no further XBP-1 splicing or ERdj4 activation could be achieved with 1NM-PP1 alone (Fig. 4, G and H) . The levels of XBP-1 splicing in cells expressing hIre1-I642G treated with 30 µM 1NM-PP1 were as high or higher than those seen in wild-type cells treated with low concentrations of DTT (Fig. 4 G) or with Tm ( Fig. 4 A) , where RIDD was clearly functioning. Note that although XBP-1 splicing appears insensitive to low concentrations of DTT in Fig. 4 G, we observed partial splicing shortly after the addition of DTT followed by rapid recovery before changes in gene expression could be reliably measured. For cells expressing wildtype hIre1, ERdj4 and RIDD appeared to be activated in parallel by low DTT concentrations, suggesting that RIDD is not limited to extreme ER stress (Fig. 4 H) . In contrast to splicing and activation of ERdj4, no change in mRNA abundance for RIDD targets was detected in cells expressing hIre1-I642G and treated with 1NM-PP1 alone, even at high concentrations, although the cells were still capable of inducing RIDD when treated with 1NM-PP1 and DTT together (Fig. 4 H) . In summary, although XBP-1 splicing and downstream transcriptional induction can be artificially induced with 1NM-PP1, RIDD cannot.