The regulation of protein synthesis by oxygen

生物体的特点是它们对刺激作出反应和适应的能力。我的研究计划集中在这些特征方面的蛋白质合成机制。蛋白质合成也被称为翻译，是遗传信息从信使核糖核酸（mRNA）流向蛋白质的基本生物过程。应激环境的变化导致翻译工作的重新聚焦，从维持基本细胞功能转向应激反应蛋白的产生。这是通过抑制初级翻译起始机制而发生的，初级翻译起始机制通过位于5'端的帽结构进入mRNA，并占翻译的>95%（帽依赖性翻译）。我的研究项目解决了细胞生物学中的一个主要问题：当帽依赖性翻译受到抑制时，细胞如何在应激期间将其mRNA翻译成蛋白质？我的重点是人类细胞中的缺氧（低氧），因为获得足够的氧气对于产生足够的ATP以维持其正常功能至关重要，特别是在构成多细胞动物的细胞中。据信，缺氧细胞在应激期间从典型的帽依赖性翻译（由帽结合蛋白eIF4E介导）转换为帽非依赖性过程以翻译选择的mRNA。我最近证明（Uniacke et al.（2012）Nature），人类细胞需要一种非经典的帽依赖性翻译途径来适应缺氧。该途径利用帽结合蛋白同源物eIF4E2，但仍存在许多未解答的问题。本提案中概述的项目旨在证明缺氧通过招募专门的翻译因子（项目1）和通过共同调控序列（项目2）的mRNA来产生专门的帽依赖性翻译反应。这些将通过免疫共沉淀、质谱、多核糖体分析、RNA测序、定点诱变和荧光素酶报告基因测定来实现。我们还将研究哪些帽依赖的翻译机器在一系列氧气条件下占主导地位，包括环境空气，生理和缺氧（项目3）。这将通过蛋白质印迹和mRNA翻译测定监测这些机制的开关活性来实现。我的长期目标是强调帽依赖性翻译在应对一系列细胞应激时的多功能性，并证明当细胞在生理氧气条件下培养时，这些专门的机器具有突出的作用。低氧eIF4E2的鉴定被CBC和National Post等媒体报道。我的研究计划将继续对自然科学产生影响，通过影响哺乳动物细胞培养实践，将氧作为一个变量，并更新当前的翻译控制模型。

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.