IRE1α Cleaves Select microRNAs During ER Stress to Derepress Translation of Proapoptotic Caspase-2
John-Paul Upton1,7,*,
Likun Wang2,6,8,*,
Dan Han2,6,8,
Eric S. Wang1,
Noelle E. Huskey2,7,
Lionel Lim2,7,
Morgan Truitt3,7,
Michael T. McManus4,5,6,
Davide Ruggero3,8,
Andrei Goga2,8,
Feroz R. Papa2,5,7,9,
, and
Scott A. Oakes1,8,
1 Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
2 Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
3 Department of Urology, University of California, San Francisco, San Francisco, CA 94143, USA.
4 Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA 94143, USA.
5 Diabetes Center, University of California, San Francisco, San Francisco, CA 94143, USA.
6 Keck Center for Noncoding RNAs, University of California, San Francisco, San Francisco, CA 94143, USA.
7 Lung Biology Center, University of California, San Francisco, San Francisco, CA 94143, USA.
8 Helen Diller Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA 94143, USA.
9 California Institute for Quantitative Biosciences, University of California, San Francisco, San Francisco, CA 94143, USA.
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Fig. 1. IRE1α is necessary and sufficient for CASP2 up-regulation. (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 fluorescence-activated cell sorting analysis of WT and DKO MEFs treated with BFA. Each data point represents the mean value ± SD from three independent experiments. (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).
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Fig. 2. The RNase activity of IRE1α up-regulates CASP2 independently of XBP1. (A) CASP2 immunoblot upon Dox induction of WT-IRE1α in T-REx-293 cells. (B) CASP2 immunoblot in T-REx 293 cells that overexpress 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.
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Fig. 3. Anti-Casp2 miRNAs decrease in IRE1α-dependent manner. (A) Quantitative polymerase chain reaction (QPCR) on polyribosome-associated Casp2 mRNA derived from Ire1α+/+ and Ire1α –/– MEFs before and after BFA treatment. (B) CASP2 Immunoblot of Ire1α+/+ MEFs treated with BFA plus or minus pretreatment with actinomycin A (ActD). QPCR of select miRNAs forms. (C) Ire1α+/+ and Ire1α –/– MEFs after BFA treatment and (D) T-REx-293 cells after overexpression 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).
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Fig. 4. 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. Each data point represents the mean value ± SD from three independent experiments. (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.
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