The endoplasmic reticulum (ER) communicates with the nucleus through the unfolded protein response (UPR), which senses accumulation of unfolded proteins in the ER lumen and leads to increased transcription of genes encoding ER-resident chaperones. As a key regulatory step in this signaling pathway, the mRNA encoding the UPR-specific transcription factor Hac1p becomes spliced by a unique mechanism that requires tRNA ligase but not the spliceosome. Splicing is initiated upon activation of Ire1p, a transmembrane kinase that lies in the ER and/or inner nuclear membrane. We show that Ire1p is a bifunctional enzyme: in addition to being a kinase, it is a site-specific endoribonuclease that cleaves HAC1 mRNA specifically at both splice junctions. The addition of purified tRNA ligase completes splicing; we therefore have reconstituted HAC1 mRNA splicing in vitro from purified components.

Phosphorylation of the α-subunit of initiation factor 2 (eIF2) controls protein synthesis by a conserved mechanism. In metazoa, distinct stress conditions activate different eIF2α kinases (PERK, PKR, GCN2, and HRI) that converge on phosphorylating a unique serine in eIF2α. This collection of signaling pathways is termed the ‘integrated stress response’ (ISR). eIF2α phosphorylation diminishes protein synthesis, while allowing preferential translation of some mRNAs. Starting with a cell-based screen for inhibitors of PERK signaling, we identified a small molecule, named ISRIB, that potently (IC50 = 5 nM) reverses the effects of eIF2α phosphorylation. ISRIB reduces the viability of cells subjected to PERK-activation by chronic endoplasmic reticulum stress. eIF2α phosphorylation is implicated in memory consolidation. Remarkably, ISRIB-treated mice display significant enhancement in spatial and fear-associated learning. Thus, memory consolidation is inherently limited by the ISR, and ISRIB releases this brake. As such, ISRIB promises to contribute to our understanding and treatment of cognitive disorders.DOI: http://dx.doi.org/10.7554/eLife.00498.001

Previously, we identified ISRIB as a potent inhibitor of the integrated stress response (ISR) and showed that ISRIB makes cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (Sidrauski et al., 2013). Here, we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation, and cognitive loss.DOI: http://dx.doi.org/10.7554/eLife.05033.001

The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers an intracellular signaling pathway, the unfolded protein response (UPR), that leads to increased transcription of genes encoding ER-resident proteins. Transcriptional activation is mediated by a dedicated transcription factor, Hac1p, whose activity is controlled by regulated splicing of its mRNA. We have identified a mutation in tRNA ligase that disrupts the UPR in the yeast Saccharomyces cerevisiae. In this mutant, splicing of HAC1 mRNA, but not tRNA, is blocked. In contrast, HAC1 mRNA splicing is not impaired in cells that are blocked in spliceosome-mediated mRNA splicing. Furthermore, the splice junctions of HAC1 mRNA do not conform to the consensus sequences of other yeast pre-mRNAs. Our results suggest that the regulated splicing of HAC1 mRNA occurs by a novel pathway, involving tRNA ligase and bypassing the spliceosome.