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 select robust RIDD candidates, we applied the following criteria to the 122 spots in clusters displaying Ire1-dependent down-regulation. First, we required the candidate RNA to be down-regulated by 1.5-fold (log 2 [DTT/untreated signal] ≤ 0.58) in at least two of the three replicates and in the mean of the three replicates. Second, to select those RNAs whose down-regulation was truly Ire1 dependent, we required that the targets be down-regulated 1.5-fold more in the hIre1 R cells compared with the Ire1 / cells and display no more than a 25% decrease in signal in response to ER stress in the Ire1 / cells. Lastly, to rule out artifacts caused by higher expression of the candidate RNAs in the hIre1 R cells in the absence of ER stress, we also required that the mean signal intensity in the presence of ER stress was lower (by 15%) in the hIre1 R cells compared with the Ire1 / cells. 26 RIDD candidates fit these criteria; these are listed in Table I. qPCR and XBP-1-splicing assays We purified RNA samples for all experiments using TRIZOL and synthesized cDNA from total RNA samples using Superscript II (Invitrogen). We then performed qPCR measurements using the primers shown in Table S1 . We measured each sample in triplicate using the Opticon (Bio-Rad Laboratories) or Realplex (Eppendorf) qPCR machines and normalized them using the signal from Rpl19, which did not change significantly relative to total RNA input concentration in any of the treatments used. We used mock cDNA samples containing no reverse transcription to ensure that the qPCR signals arose from cDNA and not from contaminating genomic DNA or other sources. To quantify the amount of XBP-1 splicing in each experiment, we amplified cDNA using primers surrounding the splice site (Table S1 ) and ran products on 2% agarose gels.