Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

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

ABSTRACT: Homeostatic control mechanisms are essentil to life. One such mechanism, the unfolded protein response (UPR) operates in all eukarytic cells to adjust protein folding capacity of the endoplasmic reticulum (ER) according to need. UPR induction in all eurkarytic cells to date involves Ire1 (kinase/endonuclease transmembrane protein) mediated a non-convential splicing of Hac1/XBP1 mRNA, encoding for a potent transcription factor. UPR induction causes a comprehensive transcriptional upregulation of the folding capacity in the ER. Here we studied the global transcriptional profile of the UPR in fission yeast. Fission yeast lacks a clear homolog of Hac1/XBP1. Instead Ire1 maintains ER homeostasis through two post-transciptional programs: selective mRNA decay and processing of Bip1 mRNA (encoding for a major HS70-familiy member in the ER) thereby stabilizing it. S. pombe cells were grown in liquid medium (YE5S) to OD600=0.25. Cells were treated with or without 2 mM DTT (Dithiothreitol, impairs disulfid bond formation) for 60 min at 30℃. Total RNA was extracted from cells using hot phenol method. PolyA+ mRNA were enriched by two sequential rounds of oligo-dT (Invitrogen) selection. rRNA depletion was performed by depletion of ll RNA smaller than 200nt (mirVana miRNA Purification Kit (Ambion) followed by two rounds of subtractive hybridization using Ribominus EukaryoteKit for RNA-seq (Invitrogen). To sequence 2',3'-cyclic phosphate cleavage products tRNA ligase was used to selectively ligate an RNA linker to all 2',3'-cyclic phosphatesin total RNA. (described in Schutz et al., 2010). Two biological repeats were performed for the following samples with different conditions and yeast strains: poly A+ mRNA sample and mRNA enriched (ribosome depletion). On replicate was performed for RNA 3' end mapping carrying a 2',3'-cyclic phosphate: 1 replicate

ORGANISM(S): Schizosaccharomyces pombe

SUBMITTER: Philipp Kimmig

PROVIDER: E-GEOD-40298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

ACCESS DATA

Publications

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

Kimmig Philipp P Diaz Marcy M Zheng Jiashun J Williams Christopher C CC Lang Alexander A Aragón Tomas T Li Hao H Walter Peter P

eLife 20121015

The unfolded protein response (UPR) monitors the protein folding capacity of the endoplasmic reticulum (ER). In all organisms analyzed to date, the UPR drives transcriptional programs that allow cells to cope with ER stress. The non-conventional splicing of Hac1 (yeasts) and XBP1 (metazoans) mRNA, encoding orthologous UPR transcription activators, is conserved and dependent on Ire1, an ER membrane-resident kinase/endoribonuclease. We found that the fission yeast Schizosaccharomyces pombe lacks b ...[more]

PMID: 23066505

Similar Datasets

Project description:The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the UPR. Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest a component of UPR induction in arv1? strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, C/EBP homologous protein (CHOP) and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis resulting in a disruption of ER integrity, one consequence of which is induction of the UPR. Yeast strains were grown to mid-logarithmic stage (A600=0.5-0.6) for RNA extraction and hybridization on Ye6100 or S98 Affymetrix gene chips. The control array (sample name C) was carried out in duplicate. The ARV1 mutant array (sample name A) was carried out in triplicate. Both ARV1 mutants Ye6100 arrays were compared to the same control Ye6100 array. Strains represent different isolates of the same genotype, mating type and genetic background (w303). Sturley lab collection (SCY) strains include SCY328 and SCY2004 for controls and SCY820 and SCY840 for ARV1 mutants.

Dataset Information

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

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure