Targeting of XBP1/HAC1 mRNA to Endoplasmic Reticulum Stress Signaling Centers

Abstract

The unfolded protein response (UPR) comprises a set of endoplasmic reticulum (ER) to nucleus signaling mechanisms that serve to adjust ER size and folding capacity to the cell needs. The core, most conserved UPR mechanism determines the non-canonical splicing of a unique mRNA encoding the transcription factor Hac1 in yeast and XBP1 in higher eukaryotes. The HAC1/XBP1 splicing is initiated by the ER stress sensor/transducer IRE1. Deficiencies in protein folding at the ER drive the clustering of IRE1 molecules into foci in the plane of the ER membrane and the activation of IRE1 kinase and RNAse cytosolic domains. Then, active IRE1 catalyzes the excision of an intron sequence localized within the HAC1/XBP1 messenger RNA, which is followed by ligation of the resulting exons by the tRNA ligase RTCB. Elimination of the intron from HAC1/XBP1 mRNA provides a translational frameshifting and yields the synthesis of Hac1/XBP1s, a key, potent UPR transcription factor. To warrant HAC1/XBP1 splicing efficiency, specific mechanisms foster the encounter between HAC1/XBP1 mRNA and IRE1 clusters at the ER. Although the IRE1 splicing mechanism is conserved from yeast to mammalian cells, different mechanisms that describe the HAC1/XBP1 mRNA transport to ER have been proposed. In yeast, it have been demonstrated that the recruitment of HAC1 mRNA to ER is mediated by an RNA stem loop element (3¿BE) located on the 3¿UTR of HAC1 mRNA and depends on the IRE1 cluster formation and on a translational repression mechanism imposed to HAC1 mRNA. Otherwise, in mammalian cells, a co-translational model explains the selective association of XBP1 mRNA to ER membranes: Ribosomes translating the unspliced XBP1 mRNA produce a hydrophobic peptide (named HR2) that, as it is synthesized by the ribosome, promotes the association of the ribosome/mRNA/nascent chain ternary complex to the cytosolic surface of the ER. Tethering of XBP1 mRNA to ER membranes facilitates its interaction with active IRE1 and the ensuing splicing. Here, we demonstrate HR2 peptide synthesis is necessary to tether XBP1 mRNA to ER, but it is dispensable for splicing under acute ER stress. In line with this notion, we have identified new determinants needed to sustain splicing in a HR2 translation-independent manner. Altogether, we propose a directional targeting mechanism to deliver XBP1 mRNA to IRE1 foci under stress conditions in mammalian cells. Additionally we deepen on the mechanism described for the HAC1 mRNA recruitment in yeast. We found a sequence on the linker domain of Ire1p that produces is necessary to recruit HAC1 mRNA. Furthermore, it was confirmed that the 3¿BE participated in targeting steps that precede HAC1 mRNA docking to Ire1p.