Direct Recruitment of eIF4GI and/DAP5 to the 5\u27 UTR of a Subset of Human mRNA Drives Their Cap-independent Translation

During unfavorable cellular conditions (e.g., tumor hypoxia, viral infection, nutrient deprivation, etc.), the canonical, cap-dependent translation initiation pathway in human cells is suppressed by sequestration of the cap-binding protein, eukaryotic initiation factor(eIF) 4E, by 4E-binding proteins. Circumvention of cap-dependent translation shutdown has been linked to tumor development and cancer progression. The stress-induced repression of cap-dependent translation has also been correlated with increased eIF4GI and its homolog, Death Associated Protein 5 (DAP5) expression levels, suggesting these factors have a role in cap-independent translation. Despite several evidence pointing towards a link upregulation of eIF4GI and /DAP5 levels during stress conditions, and the cap-independent translation of a subset of stress response mRNAs (e.g. HIF-1α, FGF-9, and p53) that drives cancer and tumor development, there has been reports showing direct quantitative measurement of these mRNAs (containing structured 5’ UTR) binding to eIF4GI/DAP5 and whether this binding drives their cap-independent translation in an eIF4GI/DAP5-dependent manner. Further, the mechanistic underpinnings of how these RNA structures within the 5’ UTRs recruits eIF4GI and/DAP5 to bypass 4EBP-mediated sequestration of eIF4E and switch from cap-dependent to cap-independent translation initiation mechanism is not well understood. Also, there are no reports showing the role of additional initiation factors in these translation mechanisms. The work described in this dissertation address this gap in knowledge by using fluorescence anisotropy-based equilibrium binding assay and luciferase-based gene expression reporter assay to provide new insights into how a subsets of human mRNAs with structured 5’ UTRs (HIF-1α, FGF-9, p53A and p53B) recruits either eIF4GI or DAP5 to mediate cap-independent mRNA translation. Results from this study provide evidence that these 5’ UTRs bind with different affinities to these proteins and that these same 5’ UTRs can promote cap-independent initiation in an eIF4GI or DAP5-dependent manner in a rabbit reticulocyte lysate depleted of eIF4GI or DAP5. We also show how translation of this subset of mRNAs with structured 5’ UTRs are either dependent on the availability of an exposed 5’ end or not, allowing us to distinguish between IRES- or CITE-mediated modes of cap-independent translation among this subset of cellular mRNAs. Further studies showed these mRNAs had distinct preferences for the additional initiation factors (eIF4A, eIF4B and eIF4E) required by these mRNAs for their cap-independent translation initiation, adding to our understanding of these different cap-independent translation mechanisms. Collectively, the quantitative binding- and in vitro translation studies obtained from this work allowed us to propose a model showing how eIF4GI/DAP5, and/or additional initiation factors are directly recruited to the structured 5’ UTRs of this subset of cellular mRNAs to facilitate a switch from cap-dependent to cap-independent modes of translation initiation