Unknown,Transcriptomics,Genomics,Proteomics

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SiRNA of human MCF10A eIF4GI depleted cells vs. controls links nutrient sensing by mTOR to cell proliferation and inhibition of autophagy

ABSTRACT: Translation initiation factors have complex functions in cells which are not yet understood. We show that depletion of initiation factor eIF4GI only modestly reduces overall protein synthesis in cells, but phenocopies nutrient-starvation or inhibition of protein kinase mTOR, a key nutrient sensor. eIF4GI depletion impairs cell proliferation, bioenergetics and mitochondrial activity, thereby promoting autophagy. Translation of mRNAs involved in cell growth, proliferation and bioenergetics were selectively inhibited by reduction of eIF4GI, whereas mRNAs encoding proliferation inhibitors and catabolic pathway factors were increased. Depletion or over-expression of other eIF4G family members did not recapitulate these results. The majority of mRNAs that were translationally impaired with eIF4GI depletion were excluded from polyribosomes due to the presence of multiple upstream open reading frames and low mRNA abundance. These results suggest that the high levels of eIF4GI observed in many breast cancers might act to specifically increase proliferation, prevent autophagy and release tumor cells from control by nutrient sensing. Global regulation of transcription and polysomal association in eIF4GI-silenced cells. Experiment Overall Design: Two sets of experiments are presented. First, overall transcriptome changes were determined for control or eIF4GI silenced cells. Next, mRNAs associated with polysomes were compared between control and eIF4GI silenced cells.

ORGANISM(S): Homo sapiens

SUBMITTER: Robert Schneider

PROVIDER: E-GEOD-11011 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project 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.

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SiRNA of human MCF10A eIF4GI depleted cells vs. controls links nutrient sensing by mTOR to cell proliferation and inhibition of autophagy

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