bims-ershed 2022-06-26 papers

Elife. 2022 Jun 22. pii: e75580. [Epub ahead of print]11

Live imaging of the co-translational recruitment of XBP1 mRNA to the ER and its processing by diffuse, non-polarized IRE1α.

Silvia Gómez-Puerta, Roberto Ferrero, Tobias Hochstoeger, Ivan Zubiri, Jeffrey Chao, Tomás Aragón, Franka Voigt.

Endoplasmic reticulum (ER) to nucleus homeostatic signaling, known as the unfolded protein response (UPR), relies on the non-canonical splicing of XBP1 mRNA. The molecular switch that initiates splicing is the oligomerization of the ER stress sensor and UPR endonuclease IRE1α (inositol-requiring enzyme 1 alpha). While IRE1α can form large clusters that have been proposed to function as XBP1 processing centers on the ER, the actual oligomeric state of active IRE1α complexes as well as the targeting mechanism that recruits XBP1 to IRE1α oligomers remains unknown. Here, we have developed a single-molecule imaging approach to monitor the recruitment of individual XBP1 transcripts to the ER surface. Using this methodology, we confirmed that stable ER association of unspliced XBP1 mRNA is established through HR2 (hydrophobic region 2)-dependent targeting and relies on active translation. In addition, we show that IRE1α-catalyzed splicing mobilizes XBP1 mRNA from the ER membrane in response to ER stress. Surprisingly, we find that XBP1 transcripts are not recruited into large IRE1α clusters, which are only observed upon overexpression of fluorescently tagged IRE1α during ER stress. Our findings support a model where ribosome-engaged, immobilized XBP1 mRNA is processed by small IRE1α assemblies that could be dynamically recruited for processing of mRNA transcripts on the ER.

Keywords: ER; IRE1; XBP1; cell biology; human; imaging; single-molecule; unfolded protein response

DOI: https://doi.org/10.7554/eLife.75580

Elife. 2022 Jun 22. pii: e74342. [Epub ahead of print]11

Endoplasmic reticulum stress activates human IRE1α through reversible assembly of inactive dimers into small oligomers.

Vladislav Belyy, Iratxe Zuazo-Gaztelu, Andrew Alamban, Avi Ashkenazi, Peter Walter.

Protein folding homeostasis in the endoplasmic reticulum (ER) is regulated by a signaling network, termed the unfolded protein response (UPR). Inositol-requiring enzyme 1 (IRE1) is an ER membrane-resident kinase/RNase that mediates signal transmission in the most evolutionarily conserved branch of the UPR. Dimerization and/or higher-order oligomerization of IRE1 are thought to be important for its activation mechanism, yet the actual oligomeric states of inactive, active, and attenuated mammalian IRE1 complexes remain unknown. We developed an automated two-color single-molecule tracking approach to dissect the oligomerization of tagged endogenous human IRE1 in live cells. In contrast to previous models, our data indicate that IRE1 exists as a constitutive homodimer at baseline and assembles into small oligomers upon ER stress. We demonstrate that the formation of inactive dimers and stress-dependent oligomers is fully governed by IRE1's lumenal domain. Phosphorylation of IRE1's kinase domain occurs more slowly than oligomerization and is retained after oligomers disassemble back into dimers. Our findings suggest that assembly of IRE1 dimers into larger oligomers specifically enables trans-autophosphorylation, which in turn drives IRE1's RNase activity.

Keywords: IRE1; UPR; cell biology; endoplasmic reticulum; human; molecular biophysics; single-molecule; stress signaling; structural biology

DOI: https://doi.org/10.7554/eLife.74342

Curr Opin Pharmacol. 2022 Jun 21. pii: S1471-4892(22)00085-6. [Epub ahead of print]65 102258

Functional role of the ER stress transducer IRE1α in CF airway epithelial inflammation.

Carla M P Ribeiro, Emily A Hull-Ryde.

Excessive and chronic airway inflammation associated with increased morbidity and mortality is a hallmark of cystic fibrosis (CF) airway disease. Previous studies underscored the role of endoplasmic reticulum (ER) signaling in CF airway inflammatory responses. In this review we discuss 1) how airway inflammation induces ER stress-triggered activation of the unfolded protein response and 2) the functional importance of the ER stress transducer inositol requiring enzyme 1α (IRE1α) in CF airway epithelial inflammatory responses. We also briefly review the current understanding of IRE1α activation and the development of small molecules aimed at modulating IRE1α kinase and RNase activities. Inhibition of IRE1α kinase and RNase may be considered as a novel therapeutic strategy to ameliorate the robust inflammatory status of CF airways.

DOI: https://doi.org/10.1016/j.coph.2022.102258

J Clin Invest. 2022 Jun 21. pii: e153519. [Epub ahead of print]

The epithelial-specific ER stress sensor ERN2/IRE1β enables host-microbiota crosstalk to affect colon goblet cell development.

Michael J Grey, Heidi De Luca, Doyle V Ward, Irini Am Kreulen, Katlynn Bugda Gwilt, Sage E Foley, Jay R Thiagarajah, Beth A McCormick, Jerrold R Turner, Wayne I Lencer.

Epithelial cells lining mucosal surfaces of the gastrointestinal and respiratory tracts uniquely express ERN2/IRE1β, a paralogue of the most evolutionarily conserved endoplasmic reticulum stress sensor ERN1/IRE1α. How ERN2 functions at the host-environment interface and why a second paralogue evolved remain incompletely understood. Using conventionally raised and germ-free Ern2-/- mice, we found that ERN2 was required for microbiota-induced goblet cell maturation and mucus barrier assembly in the colon. This occurred only after colonization of the alimentary tract with normal gut microflora, which induced Ern2 expression. ERN2 acted by splicing Xbp1 mRNA to expand ER function and prevent ER stress in goblet cells. Although ERN1 can also splice Xbp1 mRNA, it did not act redundantly to ERN2 in this context. By regulating assembly of the colon mucus layer, ERN2 further shaped the composition of the gut microbiota. Mice lacking Ern2 had a dysbiotic microbial community that failed to induce goblet cell development and increased susceptibility to colitis when transferred into germ-free wild type mice. These results show that ERN2 evolved at mucosal surfaces to mediate crosstalk between gut microbes and the colonic epithelium required for normal homeostasis and host defense.

Keywords: Cell stress; Gastroenterology; Inflammatory bowel disease

DOI: https://doi.org/10.1172/JCI153519