Quantitative dissection of protein synthesis initiation at 'omic and single mRNA scales

所有形式的生命中的细胞都依赖于蛋白质来提供结构成分以及执行几乎所有不同的功能。对于人类来说，这一过程从消化食物和从空气中提取氧气等基本过程到人们不太了解的过程，如使我们的大脑既能学习又能回忆信息。蛋白质是我们基因中编码的功能单位，依靠被称为核糖体的复杂机器来确保每次都能准确地从mRNA指令中破译每个基因序列。核糖体被特定的蛋白质合成因子引导到mRNAs上的正确位置，这些蛋白质本身由蛋白质组成，以及其他通过与核糖体和/或mRNAs相互作用而发挥作用的蛋白质。许多球员的行为方式并不完全被理解。它们共同协调了一系列复杂的过程，确保每个细胞在正确的时间和地点拥有执行一系列功能所需的蛋白质。当过程有缺陷时，这可能会导致疾病。因为蛋白质合成是所有细胞活动的基础，所以我们必须准确地理解支配这一机制的规则以及它是如何被控制的，这一点至关重要。一种描述蛋白质合成过程的通用途径已经被发现。然而，这主要源于对少量蛋白质合成的研究。来自我们实验室和其他实验室的最近证据表明，这过于简单，可能有几种不同的方法来促进核糖体向细胞中的mRNA募集。在这项建议中，我们描述了一种旨在量化蛋白质合成的第一阶段：启动过程中形成的不同复合体和中间体中存在的每个蛋白质和RNA的数量的方法。我们的方法首次结合了现代质谱学技术的使用，使用已知数量的质量标准来量化每个蛋白质组分的绝对量，以及RNA测序来量化从细胞分离的一系列可分离的翻译复合体中的RNA。此外，我们将使用新开发的使用荧光标记的显微镜方法来可视化生长细胞中蛋白质合成因子和单个mRNAs之间的相互作用。因此，我们的方法结合了互补的全球方法和定向的单个mRNA研究。这些实验将通过主动翻译细胞以及那些正在经历细胞压力的细胞来完成，这些细胞压力会迅速重新编程蛋白质合成，从而改变所研究的相互作用。拟议的工作将共同解决重要的悬而未决的问题，包括核糖体与新合成的mRNAs的第一次接触是否使用与随后在同一mRNAs上发生的启动事件相同的因素和化学计量比。无论细胞应激是否抑制大量蛋白质合成，但有利于选择性核糖体结合，都是通过改变化学计量和机制实现的。我们的单一RNA方法将提供遵循不同合成路径的机械示例mRNAs。作为一个整体，该计划将提供对“生活规则”的机械性见解。随着蛋白质合成缺陷与越来越多的人类疾病有关，所获得的见解可能对理解其机制具有广泛的价值。此外，许多生物技术应用依赖于纯化的蛋白质，从这种应用中收集到的对机制的洞察可能有助于表达系统的设计。最后，我们实施的方法论方法将更广泛地适用于其他细胞过程。

Cells in all forms of life rely on proteins to provide structural components as well as to perform almost every varied function. In people this ranges from fundamental processes such as digesting food and extracting oxygen from the air to less well understood processes such as enabling our brains to both learn and recall information. Proteins are the functional units encoded in our genes and rely on complex machines called ribosomes to ensure that each gene sequence is accurately decoded from mRNA instructions every time. Ribosomes are guided to the correct places on mRNAs by dedicated protein synthesis factors, themselves made of proteins, as well as other proteins that act by interacting with ribosomes and/or mRNAs. Many of the players act in ways that are not fully understood. Together they orchestrate a complex series of processes which ensure that every cell has the proteins needed at the right time and place to perform an array of functions. When processes are defective this can lead to disease. Because protein synthesis underpins all cellular activity it is critical that we understand precisely the rules that govern the mechanism and how it is controlled. A generic pathway describing the protein synthesis process has been uncovered. However, this stems mainly from studying the synthesis of a small number of proteins. Recent evidence from our labs and from others suggests this is overly simplistic and there may be several different ways to promote the recruitment of ribosomes to mRNAs in cells.In this proposal we describe an approach aimed to quantify the amount of each protein and RNA present in different complexes and intermediates formed during the first stage of protein synthesis: initiation. Our approach combines for the first time the use of modern mass spectrometry techniques using known amounts of mass standards to quantify absolute amounts of each protein component with RNA sequencing to quantify the RNAs within a range of separable translation complexes isolated from cells. In addition, we will use newly developed microscopy approaches using fluorescent labels to visualise interactions between protein synthesis factors and individual mRNAs in growing cells. Our approaches therefore combine complimentary global approaches with directed single mRNA studies. These experiments will be done using actively translating cells as well as those undergoing cell stresses that rapidly reprogram protein synthesis and so change the interactions studied. Together the proposed work will address important outstanding questions including whether the first engagement of ribosomes with newly made mRNAs uses the same factors and stoichiometry as subsequent initiation events on the same mRNA. Whether cell stresses that inhibit bulk protein synthesis but favour selective ribosome engagement do so with altered stoichiometry and mechanisms. Our single RNA approaches will provide mechanistic example mRNAs that follow distinct pathways to being synthesized. As a whole the program will provide mechanistic insights into the 'rules of life'. As protein synthesis defects are implicated in an increasing number of human disorders the insights gained may be broadly of value in understanding their mechanisms. In addition, many biotechnology applications rely on purified proteins and insight gleaned from this application into mechanisms may assist in the design of expression systems. Finally, the methodological approaches we implement will be more widely applicable to other cellular processes.