Translational control of cell viability downstreams of eIF4E, using polyribosome RNA

ABSTRACT: 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 4–6, <1 ribosome/transcript) and the heavy (fractions 7–10, >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.

ORGANISM(S): Mus musculus

SUBMITTER: Ola Larsson 

PROVIDER: E-GEOD-2094 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress