Functional specialization of RNP granules in RNA metabolism

虽然蛋白质是所有生物功能的主要效应者，但它们必须首先由一种称为信使RNA(信使RNA)的模板分子构成。这个过程被称为翻译，这是一个复杂的过程，在所有真核生物(动物、植物和真菌)中基本上是相同的。因此，信使核糖核酸分子的命运在决定每种特定蛋白质的产生时间、地点和数量方面至关重要。最近的工作表明，许多mRNAs是一类被称为RNA颗粒的亚细胞体的关键成分，这些颗粒被认为在mRNA的降解、储存甚至翻译过程本身中发挥着重要作用。事实上，这些含有RNA的小体或颗粒最近被证明在许多神经退行性和肌肉退行性疾病中发挥着重要作用，如脆性X智力低下、脊髓性肌肉萎缩、亨廷顿氏症和阿尔茨海默氏症。可以储存mRNA的亚细胞小体最早在30多年前被发现，现在已知有不同的味道，代表着特殊的隔室，可以分离细胞内的特定分子。与其他细胞器不同，它们没有膜，通常是由于特定蛋白质和RNA的物理性质发生变化而产生的。因此，就像水中的油滴一样，蛋白质和RNA会自我结合成液滴，形成微观上截然不同的物体。随后可以通过它们形成的条件以及结合成它们的mRNAs和蛋白质的性质来区分它们。应力颗粒(SGS)和加工体(PBS)就是两个这样的例子，它们已经成为这些生物分子凝聚体的范例。它们被认为在压力时期使mRNAs的含量合理化--储存有用的mRNAs，并可能破坏其他mRNAs。然而，我们真的不知道哪些mRNA被储存，哪些被降解，更重要的是，是什么决定了这些颗粒中mRNA的单独命运。这是了解它们在帮助细胞和生物体适应不断变化的条件方面的功能作用的先决条件。在这个项目中，我们将使用尖端的大规模技术来精确定义我们之前展示的定位到PBS的~1400个mRNAs中的大多数的分子命运。我们将使用标记技术系统地检测营养剥夺后在PBS中发现的不同mRNAs的稳定性。我们的假设是，一些mRNAs在PBS中被迅速降解，将它们从细胞中移除，而另一些mRNAs更稳定，并提供了一种来源，一旦压力消除，mRNAs可以迅速恢复蛋白质生产。突变方法将被用来定义控制PBS中mRNAs命运的顺式和反式作用因子。虽然已知在PBS中发现了部分RNA衰变机制，因此可能会促进RNA降解，但尚不清楚其他mRNAs如何在PBS中存活，基本上受到保护，使其免受降解。最后，我们将研究PBS和SGS在应激条件下如何相互作用产生功能，因为它们传统上被认为是不同的实体-尽管有许多共同的分子组成。酵母被认为是一种简单的真核生物，因此比更复杂的多细胞生物更容易研究。由于酵母中使用的所有RNA颗粒也存在于动物和植物等多细胞生物体中，我们对酵母的基础研究将指导和指导这些其他系统的研究。因此，除了对人类疾病有影响外，这项工作还将提供替代机制，以调整常用酵母和植物的工业生物技术表达系统。这项提案中的研究很可能允许在这个水平上进行优化，特别是在压力条件被证明是生物体工业化规模生长的重要因素的情况下。

Although proteins are the principal effectors of all biological function they must first be made from a template molecule known as messenger RNA (mRNA). This process, known as translation, is a complex process that is essentially identical across all eukaryotic life (animals, plants, and fungi). The fate of mRNA molecules is therefore critically important in determining when, where and how much of each specific protein is made. Recent work has shown that many mRNAs are key components of a class of subcellular bodies known as RNA granules which have been postulated to play important roles in mRNA degradation, storage, and even the translation process itself. Indeed, these RNA-containing bodies or granules have recently been shown to play important roles in many neurodegenerative and musculodegenerative diseases such as Fragile X mental retardation, spinal muscular atrophy, Huntington's and Alzheimer's. Subcellular bodies where mRNA can be stored were first discovered over 30 years ago and are now known to come in different flavours, representing specialised compartments that can segregate specific molecules inside a cell. Unlike other organelles, they have no membranes and often arise as a consequence of a change in the physical properties of specific proteins and RNAs. So rather like oil droplets in water, the proteins and RNAs self-associate into liquid droplets that form microscopically distinct bodies. They can subsequently be distinguished by the conditions under which they form, as well as the nature of the mRNAs and proteins that coalesce into them. Stress granules (SGs) and Processing bodies (PBs) are two such examples which have served as a paradigm for these biomolecular condensates. They are thought to rationalise mRNA content under times of stress- storing useful mRNAs and possibly destroying others. However, we don't really know which mRNAs are stored, which are degraded, and more importantly what determines the individual fates of the mRNAs in these granules. This is a prerequisite to understanding their functional role in helping cells and organisms to adapt to changing conditions. In this project, we will use cutting-edge large-scale technologies to precisely define the molecular fate of the majority of the ~1400 mRNAs that we have previously shown localize to PBs. We will use a labelling technique to systematically examine the stability of the different mRNAs that are found in PBs following nutrient deprivation. Our hypothesis is that that some mRNAs are rapidly degraded in PBs to remove them from cells whilst others are more stable and provide a source of mRNAs that can rapidly resume protein production once the stress is removed. Mutagenesis approaches will be used to define the cis and trans-acting factors that control the fate of mRNAs in PBs. Whilst it is known that parts of the RNA decay machinery are found in PBs and so may promote RNA degradation, it is unknown how other mRNAs can survive in PBs essentially protected against degradation. Finally, we will examine how PBs and SGs interact to engender functionality during stress conditions, as they have traditionally been considered as distinct entities - despite many molecular components in common. Yeast is considered a simple eukaryote and so is substantially easier to study than more complex multicellular organisms. Since all of the RNA granules utilised in yeast are also present in multicellular organisms such as animals and plants, our fundamental studies in yeast will guide and inform investigations in these other systems. Therefore, as well as having implications for human disease, this work will provide alternative mechanisms to tweak industrial biotechnology expression systems where yeast and plants are commonly used. The studies in this proposal may well allow optimisation at this level, especially where stress conditions prove an important factor in the industrial scale growth of an organism.