RBM39 modulates UPR signaling through alternative splicing of IRE1α/ ERN1

The unfolded protein response (UPR) preserves endoplasmic reticulum proteostasis through coordinated signaling pathways, including the IRE1α–XBP1 axis, which promotes adaptive transcriptional programs via noncanonical XBP1 mRNA splicing. However, upstream mechanisms regulating this pathway remain incompletely defined. Here, we apply CRASP-seq, a scalable RNA-coupled CRISPR screening platform, to systematically identify regulators of XBP1 splicing. We uncovered the U2 snRNP auxiliary factor RBM39 as a critical positive regulator of this process. Perturbation of RBM39 or U2 snRNP components induces alternative splicing of ERN1 , leading to exon-18 skipping and the production of an unstable transcript subject to nonsense-mediated decay, as well as a truncated IRE1α isoform that acts in a dominant-negative manner to suppress XBP1 splicing. Mechanistically, we show that heat shock reduces RBM39 functional activity and promotes ERN1 exon-18 skipping, thereby attenuating IRE1α–XBP1 signaling. Functionally, hyperactivation of this pathway is detrimental under proteotoxic stress, suggesting that exon-18 skipping serves as a stress-adaptive mechanism to limit UPR output. Together, our findings reveal a previously unrecognized regulatory axis linking the canonical splicing machinery to UPR signaling and establish alternative splicing of ERN1 as a key modulator of cellular stress responses.