The impact and regulation of eIF4A-multimerisation in establishing translational programmes

基因是构建有机体的蓝图，并定义其生物学特性。它们是由保存在细胞核中的DNA组成的。在基因表达的过程中，为了创造由基因编码的东西，DNA被复制并以一种新的形式制造成RNA。RNA中的信息由称为核糖体的分子机器读取，这些分子机器将这些信息翻译并转换为名为蛋白质的氨基酸序列。细胞内蛋白质的集合称为蛋白质组。RNA和蛋白质组是在细胞内执行功能的主要颗粒。改变细胞内的RNA可以使蛋白质组发生动态变化，从而改变细胞的功能和命运。一组基因被协同调控，并结合在一起形成一个细胞活动计划，该计划决定了哪些RNA在特定时间产生哪些蛋白质。不幸的是，由于调节这些基因表达的基因突变，这些机制可能是错误的。这种失调可能导致在错误的时间激活细胞程序，产生导致致命疾病的RNA和蛋白质。一个毁灭性的例子是癌症，当RNA导致与细胞分裂相关的蛋白质的不受控制的产生。大多数决定选择哪些RNA翻译成蛋白质的细胞过程都是通过调节翻译起始复合体eIF4F的功能来运作的。它的心脏运行着一种名为eIF4A1的蛋白质。这种蛋白质对于将RNA装载到核糖体上并启动蛋白质翻译是必不可少的。EIF4A1的活性是通过与许多其他称为辅因子的蛋白质相互作用而改变的。此外，RNA本身可以包含一些元素，这些元素可能会改变eIF4A1的功能。研究人员已经收集了eIF4A1活性如何受其辅助因子影响的信息，并开始理解eIF4A1的失调会产生致命的后果。然而，我们仍然不知道这种监管失衡是为什么以及如何建立的。为了阐明这一点，我们首先需要知道eIF4A1的功能是如何由其辅因子和RNA靶标控制的，这两个因素共同决定了这些RNA中的哪些被核糖体选择进行翻译。本研究旨在确定eIF4A1的这些模式和调控机制。人们认为eIF4A1作为单个分子具有活性，但我们最近的工作表明，eIF4A1形成蛋白质复合体，这些蛋白质复合体由一个或三个副本组装而成。我们还收集了证据表明，这些复合体具有不同的活性，并且RNA影响这些不同状态之间的分布。这就产生了RNA如何决定eIF4A1活性的假说。因此，本研究将特别关注RNA本身对eIF4A1活性的影响。为了实现这一目标，我们需要了解哪些RNA招募了哪些eIF4A1复合体，并准确地定义了RNA靶标所需的eIF4A1活性。为此，我们将分离与不同eIF4A1复合体特异结合的RNA。然后，我们将使用计算工具找出是什么将这些RNA与其他RNA区分开来。为了了解多个eIF4A1复合体是如何在RNA上发挥作用的，我们将使用最先进的显微镜来确定这些复合体的三维结构。这将确定络合物是如何形成的，并使我们能够生成不能形成这些络合物的修饰eIF4A1。我们将使用修饰的复合体来剖析它们的活性，并重建在无细胞环境中RNA如何转化为蛋白质的关键步骤，以了解它们的功能。总之，这些发现将确定eIF4A1、RNA和最终决定蛋白质翻译的辅助因素之间的关系。通过这些，我们将贡献基础知识来增加我们对eIF4A1的调节和失调以及这如何影响细胞功能的理解。此外，它可能允许设计特定的药物，从而影响特定的eIF4A1活性。

Genes are the blueprints for building an organism and define its biological properties. They are made of DNA that is kept in the nucleus of the cell. In the process of gene expression, to create what is encoded by a gene, DNA is copied and made in a new form as RNA. The information within RNAs is read by molecular machines, called ribosomes, that translate and convert this information into amino acid sequences named proteins. The collection of proteins within a cell is termed the proteome. RNA and the proteome are the main particles to carry out functions within cells. Altering the RNAs within a cell enables dynamic changes to the proteome and, hence, changes cell function and fate. Groups of genes are co-ordinately regulated and are combined to form a programme of cell activity that dictate which RNAs produce which proteins at specific times. Unfortunately, these mechanisms can be faulty due to mutations in genes that regulate the expression of these genes. Such dysregulation can lead to activation of cellular programmes at the wrong time producing RNA and proteins that cause fatal illnesses. A devastating example is cancer, when RNAs lead to uncontrolled production of proteins related to cell division.Most cellular processes that dictate which RNAs are selected for translation into protein operate by adjusting the function of the translation initiation complex eIF4F. At its hearts operates a protein called eIF4A1. This protein is essential for loading RNAs onto ribosomes and starting protein translation. eIF4A1 activity is changed by interacting with many other proteins, called cofactors. In addition, RNAs themselves can contain elements that may change which function of eIF4A1 is needed. Researchers have gathered information on how eIF4A1 activity is changed by its cofactors and are starting to understand that dysregulation of eIF4A1 has fatal outcomes. However, we still do not know why and how this dysregulation is established. To shed light on this, we first need to know how eIF4A1 function is controlled by its cofactors and RNA targets which together dictate which of these RNAs are selected for translation by the ribosome. This research proposal aims to identify these patterns and mechanisms of eIF4A1 regulation.It has been believed that eIF4A1 is active as a single molecule but our recent work revealed that eIF4A1 forms protein complexes that are assembled from one or three copies of it. We also gathered evidence that these complexes have different activities and that RNA affects the distribution between these different states. This creates the hypothesis of how RNA might dictate eIF4A1 activity. Thus, this research will focus particularly on the power of RNA itself to direct activity of eIF4A1.To achieve this goal, we need to understand which RNAs recruit which eIF4A1 complex and exactly define the eIF4A1 activity required by the RNA targets. To do this, we will isolate the RNAs that are specifically bound by different eIF4A1 complexes. We will then use computational tools to find out what discriminates these RNAs from others. To understand how the multi-eIF4A1 complexes perform their function on RNA we will determine the 3D structure of the complexes using state-of-the-art microscopes. This will identify how the complexes form and enable us to generate modified eIF4A1 that cannot form these complexes. We will use the modified complexes to dissect their activities and reconstitute key steps of how RNAs are translated into protein in a cell-free environment to understand their function. Together these findings will identify a relationship between eIF4A1, RNAs and cofactors that ultimately dictate protein translation. With this, we will contribute fundamental knowledge to increase our understanding of the regulation and dysregulation of eIF4A1 and how this affects cell function. In the further it may allow specific drugs to be designed that will affect specific eIF4A1 activities.