XBP1 mRNA Is Induced by ATF6 and Spliced by IRE1 in Response to ER Stress to Produce a Highly Active Transcription Factor

Abstract

In yeast, the transmembrane protein kinase/endoribonuclease Ire1p activated by endoplasmic reticulum stress cleaves HAC1 mRNA, leading to production of the transcription factor Hac1p that activates the unfolded protein response (UPR). In mammals, no Hac1p counterpart has yet been discovered despite the presence of Ire1p homologs in the endoplasmic reticulum. Instead, the transcription factor ATF6 specific to the mammalian UPR is regulated by intramembrane proteolysis. Here, we identified the transcription factor XBP1, a target of ATF6, as a mammalian substrate of such an unconventional mRNA splicing system and showed that only the spliced form of XBP1 can activate the UPR efficiently. Our results reveal features of the UPR conserved during evolution and clarify the relationship between IRE1- and ATF6-dependent pathways.

The unfolded protein response in fission yeast modulates stability of select mRNAs to maintain protein homeostasis

“…Activated Ire1 cleaves the XBP1 mRNA at two discrete stem-loop structures, excising a short intron. The two severed exons are then ligated to produce spliced XBP1 mRNA, which because of a frame-shift induced by the splicing event, are translated to produce active XBP1 (Yoshida et al, 2001; Calfon et al, 2002). …”

Measurement of the Unfolded Protein Response (UPR) in Monocytes

“…Cleaved ATF6 cooperates with spliced XBP-1 to induce the expression of ER chaperones, ER quality control genes, folding enzymes and ERAD (Yoshida et al, 1998;Yoshida et al, 2001).…”

Blocking of N-acetylglucosaminyltransferase V induces cellular endoplasmic reticulum stress in human hepatocarcinoma 7721 cells

“…To detect the XBP-1, 1 µM MG132 (Sigma) was used to stabilized the proteins by pretreating cells for 3 h, which was not removed when DTT was used for positive control [23]. The cellular proteins were separated and electrophoretically transferred as the previous methods [24], then probed with indicated primary antibodies (goat anti-GnT-V, goat anti-BIP, rabbit anti-XBP-1 and goat anti-GRP94 were from Santa Cruz, rabbit anti-PERK were from Cell Signaling.).…”

“…The primer pairs specific for GnT-V (5'-ggc aga aaa gca gaa cct tg-3' and 5'-agc atg cac tgg taa tga acc-3') [25], BIP (5'-ctg ggt aca ttt gat ctg act gg-3' and 5'-gca tcc tgg tgg ctt tcc agc cat tc-3') [26], β-actin (5'-acc ttc aac cca gcc atg tac-3' and 5'-ctg atc cac atc tgc tgg agg gtg g-3') [27], and XBP-1 (5'-cct tgt agt tga gaa cca gg-3' and 5'-ggg gct tgg tat ata tgt gg-3') [23] were used. The PCR reaction consisting of 1 cycle of 10 min at 94 o C, 22 cycles of 1 min at 94 o C, 1 min at 64 o C and 110 s at 72 o C, followed by 10 min at 72 o C. The products were separated on agarose gels and quantified by fluorescent imaging.…”

“…XBP-1 was a substrate of an unconventional mRNA splicing system in mammalian cells, and the splicing form of XBP-1 mRNA acted as a transcription factor which induced the expression of ER-resident molecular chaperones during ER stress [16,23,31]. To evaluate the possible role in GnT-V blocking induced ER stress, the XBP-1 was examined by RT-PCR with the primers designed according to the report by Yoshida et al [23].…”