The posttranscriptional operon theory suggests that genes are co-regulated at the level of mRNA splicing, export, localization, translational efficiency and stability—resulting in a posttranscriptional control step for each individual transcript based on sequence information in the RNA (1
). As a result, posttranscriptional control represents the critical nexus linking the transcriptome to the proteome. Posttranscriptional control is used by the cell to regulate critical life cycle events including differentiation, cell cycle transit and apoptosis (2
). Previously examined in the context of normal cellular processes (5
), derangements of posttranscriptional control are now being identified in a number of pathological states including cancer (6
), cardiovascular disorders (9
) and obesity (11
More than a decade of research has connected pathological changes in the translational machinery to a number of human malignancies including breast cancer, head and neck squamous cell carcinoma, and cancer of the prostate (12
). In cancer, the most frequently documented aberrancy in the translational apparatus is overexpression of eukaryotic translation initiation factor 4E (eIF4E), which selectively affects transport of specific transcripts (13
), increases cap-dependent translation, suppresses apoptosis and leads to malignant transformation (14
). In this regard, we have found recently that pathological activation of the translation initiation apparatus is not simply a secondary consequence of the intrinsic need of human cancer cells for increased protein synthesis, but rather is essential for expression of a transformed phenotype (16
An invariant property of malignant cells is the ability to evade apoptosis. A large repertoire of physiological regulatory cues antagonizing apoptosis activate eIF4E by signaling through the Akt-mediated kinase cascade; and recent work has directly traced the anti-apoptotic and oncogenic function of Akt to eIF4E, identifying the translation initiation apparatus as a point of convergence and integration of potentially oncogenic signals (17
). Although a few of the transcripts mediating the pro-neoplastic function of eIF4E have been deduced and validated (12
), a genome-wide analysis of the transcripts utilized by activated eIF4E to interdict apoptosis and produce a neoplastic phenotype has not been reported.
If rescue from apoptosis by eIF4E occurs through the active recruitment of ribosomes to certain transcripts and not to others, then elucidating the rules governing this process become an important goal. The posttranscriptional operon model posited by Keene and Lager (1
) suggests that by recognizing related combinations of sequence element in the 5′- and 3′-UTRs (5′- and 3′-untranslated regions) of transcripts, higher eukaryotes can co-regulate translation of mRNA subsets with a shared physiological function. Thus, by identifying genes that are translationally co-regulated by a cell under a given condition, it may be possible to identify new putative mRNA sequence elements mediating the co-regulation. There is support for the hypothesis of co-regulation dictated by RNA elements, as elements mediating specific binding to the Puf proteins have been described recently in yeast (19
). Most of the currently recognized translational regulatory elements are found in the untranslated regions of transcripts, and all known mRNA regulatory elements have been collected in the UTRsite database (20
We sought to identify those transcripts functioning in the downstream mechanism of eIF4E-mediated apoptosis resistance by combining estimates of mRNA translational efficiency and abundance to identify shared nucleotide sequences in the identified transcripts that might mediate the co-regulation, and to inform functional studies using RNA interference. The approach stratifies the mRNA population of a cell based on translational activity using polyribosome preparations, a well-defined procedure for separating transcripts based on the number of ribosomes each transcript has bound (21
), and probes the stratified transcripts with microarrays. Our global analysis of gene expression represents an advance over prior studies of posttranscriptional control in which RNA from polyribosome preparations has been used to study recruitment of ribosomes to individual transcripts of interest using northern blotting or real-time PCR (RT–PCR) (18
). In addition, although prior studies have addressed the downstream effects of activating eIF4E using polyribosome preparations in combination with microarrays; they have used rapamycin as a tool to inactivate eIF4E—an approach that also alters other key cellular functions including ribosomal biogenesis (22
) and have not systematically addressed the mechanism of eIF4E-mediated apoptotic rescue.
Here we report a genome-wide analysis of transcripts that are translationally activated when cells are rescued from a pro-apoptotic stress by overexpression of eIF4E. Our study includes a systematic UTR search identifying a consensus hairpin structure that mediates translational activation specifically under pro-apoptotic conditions, as well as siRNA-mediated knockdown experiments to assess the functional import of one prototype transcript harboring the regulatory consensus hairpin structure, osteopontin.