Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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EIF4E hypersensitive mRNA translation is characterized by long and structured untranslated regions

ABSTRACT: Combining a genetic and pharmacological approach, we modulated eIF4E activity across its physiological activity range and identified subsets of genes whose translation was either hypo- or hyper- sensitive to eIF4E activity changes. eIF4E hypersensitive genes had longer 5'UTRs with higher GC content; and longer 3'UTRs with lower GC content and a higher density of unique microRNA target sites. Proliferation related genes were enriched among eIF4E hypersensitive genes; and consistent with this, decreasing eIF4E activity inhibited cell cycle transit. Our findings provide genome wide insights into the properties of mRNAs under translational control across the physiological range of eIF4E activity. NIH 3T3 derivatives genetically altered to induce eIF4E when treated with mifepristone and a non-inducible control NIH 3T3 derivative were cultured with or without treatment with mifepristone and varying doses of a pharmacological eIF4E inhibiting agent, 4Ei-1 (0 Î¼M, 10 Î¼M, 50 Î¼M, 100 Î¼M, 200 Î¼M). Total RNA and polyribosome RNA were isolated after 4h of treatment. Three replicates of each experimental group were completed. In addition, three replicates of the inducible cell line treated with less potent analog to the inhibiting agent, 4Ei-4, were completed to probe for non-specific drug effects.

ORGANISM(S): Mus musculus

SUBMITTER: Matthew Parker

PROVIDER: E-GEOD-56072 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease. To globally address the issue of which transcripts mediate the anti-apoptotic functions of eIF4E, we used a model in which NIH 3T3 cells ectopically overexpress translation initiation factor 4E (eIF4E) resulting in an apoptosis-resistant and tumorigenic phenotype. We performed a comprehensive study of apoptosis resistance in the NIH 3T3/4E model using an array containing 15k cDNAs and ESTs (NIH 15k collection printed at The Ontario Cancer Institute). To facilitate data analysis in the two-color competitive hybridization microarray approach employed, we used a universal standard mRNA [non-stratified poly(A) selected RNA derived from NIH 3T3 cells cultured in full growth medium]. We compared signals generated from this standard to both the light (fractions 4â6, <1 ribosome/transcript) and the heavy (fractions 7â10, >1 ribosomes/transcript) polyribosome fractions of RNA from NIH 3T3 and NIH 3T3/4E cells cultured either in complete growth medium or after 16 h of serum starvation, a time point before any evidence of cytochrome c release from the mitochondria, a signature step at the apex of the cell death program. We selected the 16 h time point in order to identify genes that might govern the decision to enter the apoptotic pathway, and exclude those involved in the actual process of cell execution.

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EIF4E hypersensitive mRNA translation is characterized by long and structured untranslated regions

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