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

所有生命形式的细胞都依赖蛋白质来提供结构成分，并执行几乎所有不同的功能。在人类中，这包括从消化食物和从空气中提取氧气等基本过程到不太了解的过程，例如使我们的大脑能够学习和回忆信息。蛋白质是编码在我们基因中的功能单位，它依赖于称为核糖体的复杂机器，以确保每次都能从mRNA指令中准确解码每个基因序列。核糖体通过专门的蛋白质合成因子被引导到mRNA上的正确位置，这些蛋白质合成因子本身由蛋白质以及通过与核糖体和/或mRNA相互作用而起作用的其他蛋白质组成。许多参与者的行为方式并不完全清楚。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控制机制的规则以及它是如何被控制的。描述蛋白质合成过程的通用途径已经被发现。然而，这主要源于研究少量蛋白质的合成。最近的证据表明，从我们的实验室和其他人认为，这是过于简单化，可能有几种不同的方式来促进招聘核糖体的mRNAincell.In这个建议中，我们描述了一种方法，旨在量化每种蛋白质和RNA的量存在于不同的复合物和中间体形成的蛋白质合成的第一阶段：启动。我们的方法首次结合了现代质谱技术的使用，使用已知量的质量标准品来定量每种蛋白质组分的绝对量，并使用RNA测序来定量从细胞中分离的一系列可分离的翻译复合物内的RNA。此外，我们将使用新开发的显微镜方法，使用荧光标记来可视化生长细胞中蛋白质合成因子和单个mRNA之间的相互作用。因此，我们的方法将联合收割机互补的全球方法与定向的单个mRNA研究相结合。这些实验将使用活跃的翻译细胞以及那些经历细胞应激的细胞来完成，这些细胞应激会迅速重新编程蛋白质合成，从而改变所研究的相互作用。这些工作将共同解决重要的悬而未决的问题，包括核糖体与新产生的mRNA的第一次接合是否使用相同的因子和化学计量，作为相同mRNA的后续起始事件。是否细胞应力，抑制大量蛋白质合成，但有利于选择性核糖体接合这样做改变化学计量和机制。我们的单RNA方法将提供遵循不同途径合成的机制示例mRNA。作为一个整体，该计划将提供对“生活规则”的机械见解。由于蛋白质合成缺陷与越来越多的人类疾病有关，因此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，并且从该应用中收集到的对机制的了解可能有助于表达系统的设计。最后，我们实现的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.