2005年4月1日 · In the following report, we describe a variety of methods used to arrive at our current understanding of yeast IRE1-dependent HAC1 splicing and mammalian IRE1α-dependent XBP1 splicing, with focus on recently developed methods for detecting and measuring HAC1 or XBP1 mRNA splicing from in vitro or in vivo systems.

2014年11月6日 · Using in vitro experiments, we found that IRE1 has two different modes of action: XBP1/HAC1 is cleaved by IRE1 subunits acting cooperatively within IRE1 oligomers, whereas a single subunit of IRE1 performs RIDD without cooperativity. Furthermore, these distinct activities can be separated by complementation of catalytically inactive IRE1 RNase ...

2019年9月4日 · In ER-stressed Saccharomyces cerevisiae (hereafter simply called yeast) cells, the HAC1 mRNA is spliced by activated Ire1 molecules and then translated into a transcription-factor protein,...

Ire1p regulates synthesis of the basic leucine-zipper (bZIP)-containing transcription factor Hac1p by controlling splicing of HAC1 mRNA. Only spliced HAC1 mRNA (HAC1i) is translated, and Hac1ip activates transcription of genes that contain a …

2016年6月3日 · The inositol requiring enzyme 1 (IRE1) signaling pathway activated by the IRE1-mediated unconventional splicing of HAC1 in yeast, bZIP60 in plants and XBP1 in metazoans, is the most ancient...

2025年3月4日 · In the UPR signaling pathway, HAC1 mRNA in budding yeast 32,33,34 or XBP1 mRNA in metazoans 32,33,34 is targeted to the ER-resident protein kinase and endonuclease Ire1 32,35,36.

Here, we summarize various approaches to study the IRE1-Hac1 pathway in yeast and the homologous IRE1-XBP1 pathway in mammalian cells. We present microbiological growth assays for the UPR, reporter assays for UPR signaling, direct techniques to measure UPR activation in vivo, methods to study translation of HAC1 mRNA, and in vitro cleavage and ...

2022年2月2日 · The ability of a pre-spliced HAC1* to restore growth of ire1Δ mutants in iron-depleted medium suggested that iron limitation results in ER stress that requires Ire1-dependent HAC1 mRNA splicing. We therefore used reverse transcription PCR (RT-PCR) to detect unspliced and spliced HAC1 transcripts in the wild type and ire1 Δ mutants grown in ...

In the unfolded protein response (UPR), Ire1 activates Hac1 to coordinate the transcription of hundreds of genes to mitigate ER stress. Recent work in Caenorhabditis elegans suggests that oxidative stress inhibits this canonical Ire1 signalling pathway, activating instead an …

2014年11月6日 · IRE1, an endoplasmic reticulum (ER) kinase/RNase, splices XBP1/HAC1 to produce an active transcription factor but also cleaves ER-associated mRNA through regulated IRE1-dependent decay (RIDD). Using biochemical studies, Tam et al. demonstrate that cleavage of XBP1 and HAC1 and RIDD are distinct and separable activities.