Eukaryotic cells rely on specialized stress sensor proteins to respond to protein folding perturbations in the endoplasmic reticulum (ER). One such protein is the bifunctional kinase/endoribonuclease inositol-requiring enzyme 1 (IRE1), an ancient stress sensor whose overall structure and function are broadly conserved from yeast to humans. Upon activation, human IRE1 cleaves several RNA transcripts and initiates the only known spliceosome-independent cytosolic mRNA splicing reaction, which in turn leads to the production of the potent transcription factor XBP1s. We set out to investigate how IRE1’s RNase activity and RNA substrate specificity are regulated by the protein’s oligomeric state and phosphorylation. To this end, we developed a panel of IRE1 constructs with orthogonal control over dimerization and oligomerization and assayed the enzymatic activity of these constructs both in vitro and in live human cells. Our data converge on a model wherein phosphorylated IRE1 dimers are the principal drivers of RNA substrate recognition and cleavage, while higher order oligomerization is a transient process that serves primarily to accelerate IRE1’s autophosphorylation. Long-read transcriptomic analysis of cells expressing orthogonally controllable IRE1 identified new direct targets of IRE1’s RNase domain, expanding the known scope of the ER stress response. Finally, we observed a robust IRE1-independent increase in intron retention in the transcriptomes of cells challenged with ER stress, suggesting an unexpected link between proteostatic signaling and the mRNA splicing machinery.
Mukherjee et al. (Sun,) studied this question.