Inositol-requiring enzyme 1 (IRE1) is one of three known membrane-resident stress sensors that respond to imbalances in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a eukaryotic stress response pathway. Human IRE1 is an essential protein in both development and homeostasis that responds to ER stress by forming transient homo-oligomers. These oligomers enable trans-autophosphorylation and activation of the cytosolic RNase domain, which then initiates non-canonical splicing of the mRNA of transcription factor XBP1 into an active isoform that induces a sweeping transcriptional program. A lack of direct means of activating IRE1 has made it difficult to study independently of the other UPR sensors, leading us to engineer an IRE1 construct (called Opto-IRE1) with a light-inducible oligomerizing domain to cluster and activate the cytosolic kinase and RNase domains. To clarify the role of IRE1 within the UPR, we measured the transcriptional changes induced by Opto-IRE1 and by general ER stress in cultured human cells. With siRNA knockdown of XBP1, we also isolated the direct effects of IRE1 activity and found that XBP1-independent changes constitute a small fraction of IRE1 effects. We also created a reference-free, de novo method for identifying changes in splicing from long-read transcriptomics data. This revealed that general ER stress induced numerous changes in RNA splicing with a strong preference toward intron retention and that these changes were not IRE1 dependent. Although the UPR has been shown to regulate nonsense-mediated decay (NMD), the ER stress-induced splicing changes only partially overlapped with direct inhibition of NMD. This suggests that the UPR is using another mechanism to affect splicing. By leveraging the selectivity of Opto-IRE1, our data reveal the effects of IRE1 and the UPR on transcriptional and splicing regulation.
Smith et al. (Sun,) studied this question.