The involvement of cap-independent mRNA translation in cell fate decisions

During cell stress many biochemical processes are shut down. For example, global mRNA translation initiation is inhibited due to the disruption of the cap-dependent mRNA recruitment mechanism. One specific example of stress, apoptosis, results in activated caspases that cleave the translation initiation factor eIF4G. This cleavage disrupts cap-dependent mRNA translation initiation by removing the cap-binding domain. However, a specific subset of mRNAs can still be recruited for protein synthesis in a cap-independent manner by the residual initiation machinery. This selective recruitment of stress and apoptosis-related mRNAs promotes stress response and further induction of apoptosis. Many of these mRNAs contain internal ribosome entry sites (IRESes) that promote their enhanced translation during these conditions. Still other mRNAs have little dependence on the cap recognition mechanism. The expression of the encoded proteins, both anti- and pro-apoptotic, promote an initial period of attempted cell survival, then commitment to cell death when damage is extensive. This switch in mode of translation initiation and how it allows for selective mRNA translation is not well understood. This study focuses on the utilization of cap-independent protein synthesis in the nematode worm, C. elegans. Due to their relative genetic simplicity, while maintaining molecular pathways found in higher organisms, and semi transparency that permits direct observation of cell fate decisions, C. elegans are the fitting in vivo model in which to study changes in translational regulation and how they affect cell fate. In this study we address the translational regulation of the stress and apoptosis-related mRNAs in C. elegans: BiP (hsp-3) (hsp-4), Hif-1 (hif-1), p53 (cep-1), Bcl-2 (ced-9) and Apaf-1 (ced-4). Altered translational efficiency of these messages was observed upon depletion of cap-dependent translation and induction of apoptosis within the C. elegans gonad. Our findings suggest a physiological link between the cap-independent mechanism and the enhanced translation of hsp-3 and ced-9. This increase in the efficiency of translation may be integral to the stress response during the induction of physiological apoptosis. Further organism wide RNA-seq studies have begun to identify the entire population of mRNAs that rely highly on cap-dependent and independent translation. Development of this methodology, for detecting changes in translational efficiency on a global level, enables future follow-up studies to confirm additional mRNAs whose translation results in changes in cell fate decisions. Additionally, we carefully and specifically characterized C. elegans ced-4 mRNA, the Apaf-1 homologue, and observed that its structure and mode of translation initiation differed from its mammalian homologue. ced-4 mRNA translation illustrates one example of how selective translation in germ cells may differ from that observed in cultured mammalian cells responding to toxic treatments such as chemotherapy agents and hypoxia. Networks of translational regulation are particularly important during germ cell development. The silencing of transcription, associated with chromosome condensation during meiosis, results in protein expression patterns that are dependent translational regulation. Genetic analysis shows that loss of key translational regulators leads to the onset of germ cell tumors within the C. elegans gonad. Cell growth and mitotic mRNAs typically rely highly on cap-dependent translation initiation in mammalian cell culture. Thus, we predicted that knockdown of cap-dependent translation would decrease expression of growth and mitotic proteins and result in a reversion of tumor phenotype. However, germ cell tumor progression was not grossly affected by knockdown of cap-associated eIF4G. The inability to revert this cell fate is most likely due to other regulators of translation at work in the germ line. Reversion of tumor phenotype require knockdown of multiple regulators of translation. Overall, these results indicate an important balance between cap-dependent and -independent translation initiation during stress and the affect of this balance on germ cell fate decisions.Ph.D