The regulation of protein synthesis by oxygen

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Living organisms are characterized by their ability to respond to stimuli and adapt. My research program focuses on these characteristics with regard to the protein synthesis machinery. Also known as translation, protein synthesis is a fundamental biological process in the flow of genetic information from messenger ribonucleic acid (mRNA) into protein. Stressful environmental changes lead to a refocusing of translation efforts away from the maintenance of basic cell functions and toward the production of stress response proteins. This occurs by repressing the primary translation initiation machinery, which accesses mRNAs through their cap structure located at the 5’ end and accounts for >95% of translation (cap-dependent translation). My research program addresses a major question in cell biology: how do cells translate their mRNAs into proteins during periods of stress when cap-dependent translation is repressed? My focus is on hypoxia (low oxygen) in human cells because the acquisition of adequate oxygen is crucial to produce enough ATP for their proper functioning, especially in cells that constitute multicellular animals. It was believed that hypoxic cells switch from canonical cap-dependent translation (mediated by the cap-binding protein eIF4E) to cap-independent processes to translate select mRNAs during stress. I recently demonstrated (Uniacke et al. (2012) Nature) that a non-canonical cap-dependent translation pathway is required for human cells to adapt to hypoxia. This pathway utilizes the cap-binding protein homolog eIF4E2, but many unanswered questions remain. The projects outlined in this proposal aim to demonstrate that hypoxia creates a specialized cap-dependent translation response through the recruitment of specialized translation factors (project 1) and mRNAs via common regulatory sequences (project 2). These will be achieved through co-immunoprecipitation, mass spectrometry, polysome profiling, RNA sequencing, site-directed mutagenesis, and luciferase reporter assays. We will also investigate which cap-dependent translation machineries are dominant in a range of oxygen conditions that include ambient air, physiological, and hypoxia (project 3). This will be achieved by monitoring the activity of the on and off switches of these machineries through western blot and mRNA translation assays. My long-term goals are to highlight the versatility of cap-dependent translation in response to a range of cellular stresses, and to demonstrate that these specialized machineries have prominent roles when cells are cultured in physiological oxygen conditions. The identification of hypoxic eIF4E2 was covered by media such as the CBC and National Post. My research program will continue to have impacts on the natural sciences by influencing mammalian cell culture practices to include oxygen as a variable, and update current models of translational control.