Dynamics and specificity of RNP granules

蛋白质是生物功能的主要效应物，负责催化大多数生化反应，以及担任许多结构和调节角色。蛋白质是从一个中间分子，信使RNA（mRNA），通过一个复杂的过程，这是基本上相同的所有真核生物（动物，植物和真菌）“翻译”。越来越清楚的是，mRNA在细胞内的定位至关重要，并且可以在降解，储存和翻译过程本身中发挥作用。事实上，我们发现这些含RNA的蛋白体或颗粒在许多神经变性和肌肉变性疾病中是重要的，例如脆性X染色体智力低下、脊髓性肌萎缩、亨廷顿氏病和阿尔茨海默氏病。最近的证据表明，RNA和蛋白质的物理性质在这种颗粒的形成中很重要。这些包括形成聚集体的可能性和形成具有液体状状态的物体的能力：这些被称为“液滴”，并以类似于从醋汁混合物中分离醋和油的方式通过相分离形成。重要的是，这些液滴浓缩蛋白质和RNA，从而允许发生特异性反应，并且还播种蛋白质和RNA的更静态聚集。这些颗粒可以具有不同的功能，并在不同的条件下观察到。大体上，在细胞被转换到不利的生长条件（所谓的“应激”条件）后观察到这种颗粒，其中颗粒似乎在mRNA和蛋白质的降解或储存中起作用。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，也不知道它们是如何以及为什么首先形成的。首先，一类含有RNA的颗粒甚至存在于“正常”活跃生长的细胞中，在这些细胞中可以发生mRNA翻译成蛋白质。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，后者可以通过其沉淀能力进行纯化。在本项目中，我们将使用尖端的“omic”技术，以酵母为模型系统，精确确定含RNA颗粒的分子组成。我们将研究这些颗粒的组成如何在不同的应力前后变化，并表征不同RNA颗粒类别之间的关系以及蛋白质聚集体。最后，我们将研究一个关键问题，即决定不同蛋白质和RNA如何到达不同颗粒并最终具有不同命运的分子参数是什么。虽然酵母是一种简单的真核生物，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告知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。本提案中的研究很可能允许在此水平上进行优化，特别是在胁迫条件证明是工业发酵中的重要因素的情况下。

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.