IRE1α cleaves select microRNAs during ER stress to derepress translation of proapoptotic Caspase-2

Abstract

The endoplasmic reticulum (ER) is the primary organelle for folding and maturation of secretory and transmembrane proteins. Inability to meet protein-folding demand leads to "ER stress," and activates IRE1α, an ER transmembrane kinase-endoribonuclease (RNase). IRE1α promotes adaptation through splicing Xbp1 mRNA or apoptosis through incompletely understood mechanisms. Here, we found that sustained IRE1α RNase activation caused rapid decay of select microRNAs (miRs -17, -34a, -96, and -125b) that normally repress translation of Caspase-2 mRNA, and thus sharply elevates protein levels of this initiator protease of the mitochondrial apoptotic pathway. In cell-free systems, recombinant IRE1α endonucleolytically cleaved microRNA precursors at sites distinct from DICER. Thus, IRE1α regulates translation of a proapoptotic protein through terminating microRNA biogenesis, and noncoding RNAs are part of the ER stress response.

Fig. 1

IRE1α is necessary and sufficient for CASP2 upregulation. (A and B) Immunoblot for full length (FL) CASP2 and cleaved (Clvd) CASP2 in WT and DKO MEFs after BFA treatment. (C) Annexin-V directed FACS analysis of WT and DKO MEFs treated with BFA. (D) CASP2 immunoblot in UPR sensor deficient MEFs treated with BFA. (E) CASP2 immunoblot of Ire1α^+/+ and Ire1α ^−/− MEFs treated with tunicamycin (Tn) or thapsigargin (Tg). Each data point represents the mean value ± SD from three independent experiments.

Fig. 2

The RNase activity of IRE1α upregulates CASP2 independently of XBP1. (A) CASP2 immunoblot upon Dox-induction of WT-IRE1α in T-REx-293 cells. (B) CASP2 immunoblot in cells that over-express various IRE1α forms (C) CASP2 immunoblot in Xbp1^−/− MEFs transfected with pcDNA5-WT-IRE1α. (D) CASP2 immunoblot in T-REx-293 cells before and after Dox-induction of XBP1s.

Fig. 3

Anti-Casp2 miRNAs decrease in IRE1α-dependent manner. (A) qPCR on poly-ribosome associated Casp2 mRNA derived from Ire1α^+/+ and Ire1α ^−/− MEFs before and after BFA treatment (B) CASP2 Immunoblot of Ire1α^+/+ MEFs treated with BFA plus/minus pre-treatment with actinomycin A (ActD). qPCR of select miRNAs from (C) Ire1α^+/+ and Ire1α ^−/− MEFs after BFA treatment and (D) T-REx-293 cells after over-expression of WT-IRE1α or 1NM-PP1 activation of IRE1α (I642G). Each data point represents the mean value ± SD from three independent experiments. Asterisks indicate a statistically significant change from the vehicle treated controls (p<0.05).

Fig. 4

WT-IRE1α directly cleaves pre-miR-17. CASP2 immunoblot of T-REx-293 cells transfected with indicated (A) anti-miRNAs or (B) miRNA mimics after Dox-induction of WT-IRE1α. (C) qPCR of pri-, pre-, and mature miR-17 after IRE1α activation in WT-IRE1α T-REx-293 cells. (D) Radioblot of ³²P-labeled pre-miR-17 digestion products after incubation with indicated recombinant IRE1α proteins. (E) Mapping of IRE1α cleavage sites in pre-miR-17. (F) Illustration of the IRE1α cleavage sites within pre-miR-17. Each data point represents the mean value ± SD from three independent experiments.