Summary Protein misfolding stimulates a signaling pathway involving noncoding RNAs to promote cell death. 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, -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.

SUMMARY Depending on endoplasmic reticulum (ER) stress levels, the ER transmembrane multi-domain protein IRE1α promotes either adaptation or apoptosis. Unfolded ER proteins cause IRE1α lumenal domain homo-oligomerization, inducing trans auto-phosphorylation that further drives homo-oligomerization of its cytosolic kinase/ endoribonuclease (RNase) domains to activate mRNA splicing of adaptive XBP1 transcription factor. However, under high/chronic ER stress, IRE1α surpasses an oligomerization threshold that expands RNase substrate repertoire to many ER-localized mRNAs, leading to apoptosis. To modulate these effects, we developed ATP-competitive IRE1α Kinase Inhibiting RNase Attenuators—KIRAs—that allosterically inhibit IRE1α’s RNase by breaking oligomers. One optimized KIRA, KIRA6, inhibits IRE1α in vivo and promotes cell survival under ER stress. Intravitreally, KIRA6 preserves photoreceptor functional viability in rat models of ER stress-induced retinal degeneration. Systemically, KIRA6 preserves pancreatic β-cells, increases insulin, and reduces hyperglycemia in Akita diabetic mice. Thus, IRE1α powerfully controls cell fate, but can itself be controlled with small molecules to reduce cell degeneration.

SUMMARY The intrinsic (mitochondrial) apoptotic pathway is a conserved cell death program crucial for eliminating superfluous, damaged, or incorrectly specified cells [1], and the multi-domain pro-death BCL-2 family proteins BAX and BAK are required for its activation [2, 3]. In response to internal damage or developmental signals, BAX and/or BAK permeabilize the mitochondrial outer membrane [4, 5], resulting in cytochrome c release and activation of effector caspases such as Caspase-3 (Casp3) [6]. While the mitochondrial apoptotic pathway plays a critical role during late embryonic development in mammals [3], its role during early development remains controversial [7, 8]. Here, we show that Bax−/−Bak−/− murine embryonic stem cells (ESCs) display defects during the exit from pluripotency, both in culture and during teratoma formation. Specifically, we find that when ESCs are stimulated to differentiate, a subpopulation fails to do so and instead upregulates FAS in a p53-dependent manner to trigger Bax/Bak-dependent apoptosis. Blocking this apoptotic pathway prevents the removal of these poorly-differentiated cells, resulting in the retention of cells that have not exited pluripotency. Taken together, our results provide further evidence for heterogeneity in the potential of ESCs to successfully differentiate, and reveal a novel role for apoptosis in promoting efficient ESC differentiation by culling cells that are slow to exit pluripotency.