The function and regulation of translationally active RNA granules

几乎所有的生命形式都需要产生能量的能力和一种将基因中的信息转化为不同氨基酸链的机制，这种氨基酸链被称为蛋白质。蛋白质是生物功能的主要效应器，负责催化大多数生化反应，包括产生能量的反应和蛋白质生产本身所需的反应。基因从中间分子信使RNA （mRNA）翻译成蛋白质，这一过程在所有真核细胞（动物、植物和真菌）中都是高度相似的。蛋白质和mrna都可以在细胞中定位，从而产生特定蛋白质的局部浓度，这在细胞带的空间发育中起着关键作用，例如神经细胞上的长突起或肠细胞上的膜突起。mRNA定位到这些位点涉及颗粒，颗粒中含有处于惰性、抑制状态的mRNA。惰性mrna也可以在细胞逆境中被定位，其中已经确定了两种不同类型的颗粒，“应激颗粒”和“p体”。这些颗粒被认为在储存有用的mrna和破坏多余的mrna中发挥作用。此外，它们的分布还与人类疾病，特别是大脑和肌肉疾病有关，并在多细胞动物的发育，特别是胚胎的发育中起着根本作用。我们使用简单的单细胞生物，啤酒酵母，作为模型来研究这些基本过程。在酵母中，mRNA定位于定义的极化区域和p -体或应激颗粒已被广泛研究，以揭示控制蛋白质合成的关键原理。酵母因其相对简单的基因组、与人类细胞的相似性以及基因容易突变、删除或以某种方式标记而成为此类研究的范例。事实上，整个酵母菌株的收集已经被构建，所有的酵母菌基因都被系统地删除或标记，这有助于一系列无偏倚的筛选，在这些筛选中，单个菌株被测试活性变化。我们最近的研究在酵母中发现了一个特别新颖的发现-即使在活跃生长的细胞中，mRNA颗粒中也存在编码参与能量产生和蛋白质合成的非局部蛋白质的mRNA。与大多数mRNA定位事件相反，这些mRNA不是惰性的，相反，它们在这些颗粒中被翻译成蛋白质。我们已经在几个方向上扩展了这项工作。值得注意的是，我们发现两种途径中几乎所有mRNA都在颗粒中共定位和翻译。我们利用酵母作为一种系统来进行公正的筛选，并确定对这些颗粒重要的基因。这些基因编码的蛋白质具有RNA结合和蛋白质折叠等功能。我们也采取了假设驱动的方法来确定mRNA定位到颗粒的关键决定因素。因此，我们表明指示mRNA产生的DNA序列称为启动子，以及mRNA的化学修饰在决定特定mRNA颗粒的命运方面是重要的。在这个项目中，我们将破译mRNA选择颗粒的精确决定因素以及这对细胞生理的影响。我们将研究mrna如何被标记为颗粒状命运，并研究破译这些标记的蛋白质。我们还将研究聚焦于能量和蛋白质生产的定位的功能原理，特别是在细胞分裂期间。这些基础研究将指导和指导包括人类在内的其他系统的研究，并提供替代机制来调整酵母通常使用的工业生物技术系统。本提案中的研究可能很好地允许在这个水平上进行优化，特别是对于多蛋白生化途径。

Almost all life forms require the capacity to produce energy and a mechanism to convert the information in genes into chains of different amino acids called proteins. Proteins are the principal effectors of biological function, responsible for catalysing most biochemical reactions including those that produce energy and those required for protein production itself. Genes are translated into protein from an intermediate molecule, messenger RNA (mRNA), in a process that is highly similar across all eukaryotic cells (animals, plants and fungi). Both proteins and mRNAs can be localised in cells to allow the generation of local concentrations of specific proteins, and this plays critical roles in the spatial development of cellular zones such as long projections on nerve cells or membrane protrusions on gut cells. mRNA localisation to such sites involves granules which contain the mRNAs in an inert, repressed state. Inert mRNAs can also become localised during cellular adversity, where two different classes of granule have been identified, 'stress granules' and 'P-bodies'. These granules are thought to play roles in both the storage of useful and destruction of surplus mRNAs. Additionally, their deployment has also been linked to human disease, especially in diseases of the brain and muscles, as well as fundamental roles in the development of multicellular animals, especially development of the embryo. We use the simple single-celled organism, brewer's yeast, as a model to study these fundamental processes. mRNA localisation both to defined polarised regions and to P-bodies or stress granules has been widely studied in yeast to uncover key principles that control protein synthesis. Yeast has served as a paradigm in such studies owing to its relatively simple genome, its level of similarity to human cells and the ease with which genes can be mutated, deleted or tagged in some way. In fact whole yeast strains collections have been constructed where all of the yeast genes have been systematically deleted or tagged, and these have facilitated a range of unbiased screens, where individual strains are tested for activity changes.Our recent studies have uncovered a particularly novel finding in yeast- mRNAs encoding non-localised proteins involved in energy generation and protein synthesis are present in mRNA granules even in actively growing cells. Counter to most mRNA localisation events, these mRNAs are not inert, instead they are translated into protein in these granules. We have extended this work in a number of directions. Remarkably, we have found that almost every mRNA across the two pathways is co-localised to and translated in granules. We have taken advantage of yeast as a system to perform unbiased screens and identify genes that are important for these granules. These genes encode proteins with functions such as RNA binding and protein folding. We have also taken hypothesis driven approaches to identify key determinants involved in the localisation of mRNA to granules. As such, we show that DNA sequences dictating mRNA production called promoters, as well as the chemical modification of mRNA are important in determining the fate of specific mRNAs to granules. In this project, we will decipher the precise determinants of mRNA selection to granules and how this impacts on the physiology of cells. We will examine how the mRNAs are marked for a granular fate and investigate the proteins that decipher these marks. We will also investigate the functional rationale for the localisation focussing on energy and protein production, especially during cell division. These fundamental studies will guide and inform studies in other systems including human, as well as provide alternative mechanisms to tweak industrial biotechnology systems where yeast is commonly used. The studies in this proposal may well allow optimisation at this level, especially for multi-protein biochemical pathways.