Abstract
Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs—degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.
Highlights
Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR)
By isolating homotypic complexes of the human IRE1α kinase-endoribonuclease (IRE1-KR) module in monomeric, dimeric, or oligomeric form, we demonstrate that regulated IRE1-dependent decay (RIDD) and RIDD lacking endomotif” (RIDDLE) reside in two distinct RNase modalities: endomotif-directed cleavage, minimally requiring IRE1α dimers; and endomotif-independent cleavage, necessitating phosphooligomers
We identified 54 mRNAs as potential RIDD substrates: these displayed an IRE1α-dependent, ER stress-induced decrease in abundance of at least 1.4-fold as measured by RNAseq, without a corresponding decline in transcription as measured by global nuclear run-on sequencing (GROseq) (Supplementary Table 1)
Summary
Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Excess folding demand leads to ER accumulation of misfolded proteins, causing ER stress. This engages an intracellular signaling network, dubbed the unfolded-protein response (UPR), which aims to reestablish homeostasis[1,2,3]. Unfolded-protein sensing by the lumenal domain drives IRE1α homo-oligomerization, kinase-mediated trans-autophosphorylation, and endoribonuclease activation[19,20,21,22,23,24]. CNG|CAGN within the loop, with scission between G and C in the third and fourth positions[34,35]
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