Dynamics and specificity of RNP granules

蛋白质是生物功能的主要效应者，负责催化大多数生化反应，并发挥许多结构和调节作用。蛋白质是通过一个复杂的过程从信使RNA(信使RNA)“翻译”出来的，这个复杂的过程在所有真核生物(动物、植物和真菌)中基本上都是相同的。越来越清楚的是，mRNA在细胞内的定位是至关重要的，可以在降解、储存和翻译过程中发挥作用。事实上，我们正在发现，这些含有RNA的蛋白质小体或颗粒在许多神经退行性和肌肉退行性疾病中非常重要，如脆性X智力低下、脊髓性肌萎缩症、亨廷顿氏症和阿尔茨海默氏症。最近的证据表明，RNA和蛋白质的物理性质在这些颗粒的形成中非常重要。它们包括形成聚集体的可能性和形成具有类似液体状态的物体的能力：这些被称为“液滴”，以类似于从醋汁混合物中分离食醋和油的方式通过相分离形成。关键的是，这些液滴浓缩了蛋白质和RNA，允许发生特定的反应，也播下了蛋白质和RNA更静态的聚集。最近，在识别不同的含有RNA的颗粒方面出现了巨大的热潮。这些颗粒可以具有不同的功能，并在不同的条件下进行观察。大体上，这样的颗粒是在细胞切换到不利的生长条件(所谓的‘应激’条件)后观察到的，在这种条件下，颗粒似乎在降解或储存mRNA和蛋白质方面发挥作用。然而，我们并不真正知道哪些蛋白质和RNA对哪些颗粒类型的形成是重要的，也不知道它们最初是如何形成的和为什么形成的。我们最近的研究发现了两个新的发现。首先，即使在“正常”生长活跃的细胞中也存在一类含有颗粒的RNA，在这些细胞中，mRNA可以转化为蛋白质。在第二项研究中，我们发现，应激后形成的RNA颗粒与蛋白质聚集体非常相似，蛋白质聚集体可以凭借其沉淀能力进行纯化。在这个项目中，我们将使用尖端的组学技术，以酵母为模型系统，精确定义含有RNA的颗粒的分子组成。我们将研究这些颗粒在不同压力前后的组成是如何变化的，并表征不同的RNA颗粒类别之间的相互关系以及与蛋白质聚集体的关系。最后，我们将研究一个关键问题，那就是什么分子参数决定了不同的蛋白质和RNA如何到达不同的颗粒，最终产生不同的脂肪。虽然酵母是一个简单的真核生物，但酵母中使用的所有RNA颗粒也存在于高等细胞中。因此，我们对酵母的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常用酵母的工业生物技术表达系统。这项建议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工业发酵中的一个重要因素的情况下。

Proteins are the principal effectors of biological function, responsible for catalyzing most biochemical reactions, as well as serving numerous structural and regulatory roles. Proteins are 'translated' from an intermediate molecule, messenger RNA (mRNA), by a complex process that is essentially identical across all eukaryotic life (animals, plants and fungi). It is becoming increasingly clear that the localization of mRNA within the cell is critically important and can play roles in the degradation, storage and the translation process itself. Indeed, we are discovering that these RNA-containing protein bodies or granules are important in a number of neurodegenerative and musculodegenerative diseases such as Fragile X mental retardation, spinal muscular atrophy, Huntington's and Alzheimer's. Recent evidence suggests that the physical properties of the RNA and protein are important in the formation of such granules. These include the likelihood to form aggregates and the capacity to form bodies that have a liquid-like state: these have been called 'liquid droplets' and form by phase separation in a manner analogous to the separation of vinegar and oil from a vinaigrette mix. Critically, these liquid droplets concentrate protein and RNA allowing specific reactions to occur and also seeding the more static aggregation of proteins and RNA.Recently, there has been a huge upsurge in the identification of different RNA containing granules. These granules can have different functions and are observed under different conditions. In the main, such granules have been observed after cells have been switched to unfavourable growth conditions (so-called 'stress' conditions) where the granules appear to play roles in the degradation or storage of mRNA and protein. However, we don't really know which proteins and RNAs are important for the formation of which granule types, nor how and why they form in the first place.Our recent studies have uncovered two novel findings. Firstly, a class of RNA containing granules exist even in 'normal' actively growing cells where mRNA translation into protein can occur. In a second study, we have found that RNA granules formed after stress are remarkably similar to protein aggregates that can be purified by virtue of their capacity to sediment. In this project, we will use cutting-edge 'omic technologies to precisely define the molecular composition of RNA-containing granules using yeast as a model system. We will examine how the composition of these granules changes before and after different stresses and characterize the relationship between the different RNA granule classes to each other and to protein aggregates. Finally, we will examine a key question, which is what are the molecular parameters determining how different proteins and RNAs arrive in different granules to ultimately have distinct fates.Although yeast is a simple eukaryote, all of the RNA granules utilized in yeast are also present in higher cells. Hence, our fundamental studies in yeast will guide and inform studies in other systems including human, as well as provide alternative mechanisms to tweak industrial biotechnology expression systems where yeast is commonly used. The studies in this proposal may well allow optimization at this level, especially where stress conditions prove an important factor in the industrial fermentation.