Optogenetic Clustering of Human IRE1 Reveals Differential Regulation of Transcription and mRNA Splice Isoform Abundance by the UPR

Inositol-requiring enzyme 1 (IRE1) is one of three known sensor proteins that respond to homeostatic perturbations in the metazoan endoplasmic reticulum. The three sensors collectively initiate an intertwined signaling network called the Unfolded Protein Response (UPR). Although IRE1 plays pivotal roles in human health and development, understanding its specific contributions to the UPR remains a challenge due to signaling crosstalk from the other two stress sensors. To overcome this problem, we engineered a light-activatable version of IRE1 and probed the transcriptomic effects of IRE1 activity in isolation from the other branches of the UPR. We demonstrate that 1) oligomerization alone is sufficient to activate IRE1 in human cells, 2) IRE1’s transcriptional response evolves substantially under prolonged activation, and 3) the UPR induces major changes in mRNA splice isoform abundance in an IRE1-independent manner. Our data reveal previously unknown targets of IRE1 transcriptional regulation and direct degradation. Additionally, the tools developed here will be broadly applicable for precise dissection of signaling networks in diverse cell types, tissues, and organisms. RNAseq of U-2 OS cells expressing either WT IRE1 or Opto-IRE1 (a light-inducible construct of IRE1 with an appended CRY2-clust domain) treated with light, tunicamycin, anti-XBP1 siRNA, or NMDI inhibitor 11j to dissect the effects of IRE1 activation.