Comment[ArrayExpressAccession] E-GEOD-54581 MAGE-TAB Version 1.1 Public Release Date 2014-06-02 Investigation Title Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha Comment[Submitted Name] Selective mRNA translation during eIF2 phosphorylation induces expression of IBTKalpha Experiment Description Disruption of protein folding in the endoplasmic reticulum triggers the Unfolded Protein Response (UPR), a transcriptional and translational control network designed to restore protein homeostasis. Central to the UPR is PERK phosphorylation of the alpha subunit of eIF2 (eIF2~P), which represses global translation coincident with preferential translation of mRNAs, such as ATF4 and CHOP, that serve to implement the UPR transcriptional regulation. In this study, we used sucrose gradient ultracentrifugation and a genome-wide microarray approach to measure changes in mRNA translation during ER stress. Our analysis suggests that translational efficiencies vary across a broad range during ER stress, with the majority of transcripts being either repressed or resistant to eIF2~P, while a notable cohort of key regulators are subject to preferential translation. From this latter group, we identify IBTKa as being subject to both translation and transcriptional induction during eIF2~P in both cell lines and a mouse model of ER stress. Translational regulation of IBTKalpha mRNA involves the stress-induced relief of two inhibitory uORFs in the 5'-leader of the transcript. Depletion of IBTKalpha by shRNA reduced viability of cultured cells coincident with increased caspase 3/7 cleavage, suggesting that IBTKalpha is a key regulator in determining cell fate during the UPR. We used a genome-wide microarray approach to determine how individual mRNAs were differentially translated during endoplasmic reticulum stress. A microarray analysis from our laboratory identified gene transcripts suggested to be under translation control in mouse embryonic fibroblast (MEF) cells following a 6 hour treatment with thapsigargin, a potent inducer of ER stress, or no stress. The mRNAs were separated by sucrose gradient analyses to yield three fractions, those transcripts associated with large polysomes (≥4 ribosomes per mRNA), those associated with monosome, disomes, or trisomes, and those fractionated at the top of the gradient with free ribosomes. RNA was extracted from sucrose gradients corresponding to these fractions and hybridized on Affymetrix microarrays. In parallel, we also measured total levels for each gene transcript in the presence or absence of thapsigargin treatment to address transcription regulation coincident with translational control. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples. Term Source Name ArrayExpress EFO Term Source File http://www.ebi.ac.uk/arrayexpress/ http://www.ebi.ac.uk/efo/efo.owl Person Last Name Wek Baird McClintick Wek Person First Name Ronald Thomas Jeanette Ronald Person Mid Initials C D N C Person Email rwek@iu.edu Person Affiliation Indiana University School of Medicine Person Phone 317-274-0549 Person Address Biochemistry & Molecular Biology, Indiana University School of Medicine, 6238 Brokenhurst Road, Indianapolis, Indiana, USA Person Roles submitter Protocol Name P-GSE54581-1 P-GSE54581-5 P-GSE54581-6 P-GSE54581-2 P-GSE54581-3 P-GSE54581-4 P-GSE54581-7 Protocol Description MAS5 data was generated using Affymetrix GeneChip Operating Software (GCOS) and scaling = 1 so that no scaling was done. Arrays were scaled using the 3' and M poly-A bacterial spike-in probe set values as stated in Sampth, et al.: PMID 18462695. Please note that the treatment plus fractionation based on association with different numbers of ribosomes did yield different populations of mRNAs, which resulted in considerable variation in normalized data across the samples. ID_REF = VALUE = MAS5 algorithm in Affymetrix GCOS as scaled using the bacterial poly-A spike-ins. Detection = RNA preparations were labeled using the standard Affymetrix protocol for 3'-IVT arrays (Affymetrix, Santa Clara, CA) using 2ug of RNA for all samples. The poly A spike-in was omitted because they were spiked into the riobosomal fractions before RNA extraction to be used for normalization later. Labeled cRNA was hybridized for 17 hours to the Affymetrix Mouse Genome 430 2.0 Array. MEF cells were treated with 1 uM thapsigargin for 6 hours, or without treatment, and rinsed twice with chilled PBS before Trizol extraction. For polysome analysis, both treatment groups were also subjected to a 10 minute (50 ug/mL) cyclohexidime treatment prior to sucrose gradient ultracentrifugation and RNA extraction. Mouse embryonic fibroblast (MEF) cells were grown in DMEM media supplemented with mM non-essential amino acids, 10% fetal bovine serum, 100 units/mL penicillin, and 100 ug/mL streptomycin at 37C. Cell lysates were subjected to sucrose gradient centrifugation and polysome fractionation. A total of 14 fractions were collected from the top of the gradients into cold microfuge tubes and immediately placed on ice. Each fraction was adjusted to 0.5% SDS, and fractions were combined to form three pools as follows: fractions 1-4, 5-8, 9-14 were combined as pools 1, 2, and 3, respectively. In parallel, total RNA was isolated from unfractionated lysates for analysis of total gene transcript levels. Synthetic Poly(A) luciferase RNA (10ng/mL) (Promega), along with a bacterial spike-in control RNA (Affymetrix), were added to each gradient fraction pool. RNA was precipitated at -70C with 2.5 volumes 100% ethanol and purified using QIAGEN RNeasy midi-columns. Arrays were scanned using GCOS. Protocol Type normalization data transformation protocol labelling protocol hybridization protocol sample treatment protocol growth protocol nucleic acid extraction protocol array scanning protocol Experimental Factor Name RNA FRACTION TREATED WITH MOLECULE SUBTYPE REPLICATE Experimental Factor Type rna fraction treated with molecule subtype replicate PubMed ID 24648495 Comment[SecondaryAccession] GSE54581 Comment[GEOReleaseDate] 2014-06-02 Comment[ArrayExpressSubmissionDate] 2014-01-31 Comment[GEOLastUpdateDate] 2014-06-02 Comment[AEExperimentType] transcription profiling by array SDRF File E-GEOD-54581.sdrf.txt