Messenger RNA targeting to endoplasmic reticulum stress signalling sites

Abstract

Deficiencies in the protein folding capacity of the endoplasmic reticulum (ER) in all eucaryotic cells lead to ER stress and triggers the unfolded protein response (UPR)1–3. ER stress is sensed by Ire1, a transmembrane kinase/endoribonuclease, which initiates the non-conventional splicing of the mRNA encoding a key transcription activator, Hac1 in yeast or XBP-1 in metazoans. In the absence of ER stress, ribosomes are stalled on unspliced HAC1 mRNA. The translational control is imposed by a base pairing interaction between the HAC1 intron and the HAC1 5′ untranslated region (5′UTR)4. After excision of the intron, tRNA ligase joins the severed exons5,6, lifting the translational block and allowing synthesis of Hac1 from the spliced HAC1 mRNA to ensue4. Hac1 in turn drives the UPR gene expression program comprising 7–8% of the yeast genome7 to counteract ER stress. We show here that upon activation, Ire1 molecules cluster in the ER membrane into discrete foci of higher-order oligomers, to which unspliced HAC1 mRNA is recruited by means of a conserved bipartite targeting element contained in the 3′ untranslated region (3′UTR). Disruption of either Ire1 clustering or of HAC1 mRNA recruitment impairs UPR signaling. The HAC1 3′UTR element is sufficient to target other mRNAs to Ire1 foci, as long as their translation is repressed. Translational repression afforded by the intron fulfills this requirement for HAC1 mRNA. Recruitment of mRNA to signaling centers provides a new paradigm for the control of eukaryotic gene expression.

Multiple Mechanisms of Unfolded Protein Response–Induced Cell Death

“…22,23 Interestingly, Xbp1 mRNA splicing only occurs during the acute phase. 23e25 During the chronic phase of ER stress, IRE1's RNase substrate specificity is altered to cleave primarily ER-targeted mRNAs in a process termed regulated IRE1-dependent mRNA decay (RIDD) 26e28 ( Figure 2).…”

“…The IRE1 oligomeric clusters formed by ER stress also act as molecular scaffolds to recruit other proteins and nucleate the formation of stress signal transduction sites at the ER lipid bilayer 22,23 (Figure 2). For example, TRAF2 adaptor protein binds to IRE1 as well as the ASK1 MAPKKK to activate cytosolic signaling kinases, such as C-Jun N-terminal kinase, p38, and extracellular signaleregulated kinase during ER stress 36,37 (Figure 2).…”

Approaches to imaging unfolded secretory protein stress in living cells

“…An Ire1 mutant unable to dimerize fails to activate the UPR [70]. Ire1 oligomers in yeast cells can be detected by immunofluorescence [25, 71]. To visualize Ire1 oligomers in living intact cells, it is possible to fluorescently tag Ire1 with FPs, such as GFP [24, 71].…”

“…Ire1 oligomers in yeast cells can be detected by immunofluorescence [25, 71]. To visualize Ire1 oligomers in living intact cells, it is possible to fluorescently tag Ire1 with FPs, such as GFP [24, 71]. However, to conserve all of the Ire1 protein functions, the FP needs to be inserted into the Ire1 linker domain.…”

“…However, to conserve all of the Ire1 protein functions, the FP needs to be inserted into the Ire1 linker domain. Indeed, N or C terminus-GFP-tagged Ire1 reportedly cannot activate the UPR [71]. Upon treatment with ER stressors, Ire1-GFP displays oligomer formation that can be quantified in live cells using fluorescence microscopy [24, 71, 72].…”