The regulation of protein synthesis by oxygen

有生命的有机体的特征是它们对刺激的反应和适应的能力。我的研究项目集中在蛋白质合成机制的这些特征上。蛋白质合成也被称为翻译，是信使核糖核酸(MRNA)向蛋白质传递遗传信息的基本生物过程。应激环境的变化导致翻译工作从维持基本细胞功能转向生产应激反应蛋白。这是通过抑制初级翻译启动机制发生的，该机制通过位于5‘端的帽结构访问mRNAs，占翻译的95%(取决于帽的翻译)。我的研究项目解决了细胞生物学中的一个主要问题：当依赖帽子的翻译受到抑制时，细胞如何在应激期间将其mRNAs翻译成蛋白质？我的重点是人类细胞的缺氧(低氧)，因为获得足够的氧气对于产生足够的ATP以使其正常运作至关重要，特别是在构成多细胞动物的细胞中。人们认为，在应激过程中，低氧细胞从典型的帽依赖翻译(由帽结合蛋白eIF4E介导)转换为帽非依赖过程来翻译特定的mRNAs。我最近演示了(Uniacke等人(2012)自然)，人类细胞需要一个非规范的帽依赖的翻译途径来适应缺氧。这一途径利用了帽子结合蛋白同系物EIF4E2，但仍有许多未回答的问题。本提案中概述的项目旨在证明，低氧通过招募专门的翻译因子(项目1)和经由共同调控序列的mRNAs(项目2)来产生专门的帽子依赖的翻译反应。这些将通过免疫共沉淀、质谱学、多聚体图谱、RNA测序、定点突变和荧光素酶报告分析来实现。我们还将调查哪些帽子依赖的翻译机制在包括环境空气、生理和低氧在内的一系列氧气条件下占主导地位(项目3)。这将通过蛋白质印迹和信使核糖核酸翻译分析监测这些机器的开关活动来实现。我的长期目标是强调应对一系列细胞压力时依赖帽子的翻译的多功能性，并证明当细胞在生理氧气条件下培养时，这些专门的机械具有显著的作用。CBC和National Post等媒体报道了EIF4E2缺氧症的鉴定。我的研究计划将继续对自然科学产生影响，影响哺乳动物细胞培养实践，将氧气作为一个变量，并更新目前的翻译控制模型。

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.