Residue-by-residue details of FUS protein phase separation and aggregation

项目摘要 RNA结合蛋白是许多大复合物的基本组成部分， 包括转录、剪接和DNA修复的过程。许多RNA结合蛋白具有 基于对正常RNA至关重要的低复杂性序列特征，预测其会发生无序- 加工功能，而且还驱动各种神经退行性疾病中的异常蛋白质组装， 癌的这些无序结构域的分子相互作用和功能作用仍然不完全 特征，尤其是在疾病的背景下。融合肉瘤（FUS）是29个人类肿瘤之一， RNA结合蛋白，包含一个基本的无序的低复杂性结构域（LC）， 低电荷残基组成和高频率的芳香族氨基酸以及几个RGG基序 地区尽管存在紊乱，但这些结构域被认为通过以下方式促进正常RNA代谢中的相互作用： 形成动态关联，从而实现可调的、可逆的空间聚类。然而，过度的自我- FUS无序结构域之间的关联被认为导致病理性神经元损伤的形成。 肌萎缩侧索硬化症（ALS）和额颞叶痴呆（FTD）亚型中的内含物，不可逆 缺乏有效治疗的神经退行性疾病。此外，FUS无序结构域与 几个DNA结合域通过染色体易位导致基因表达失控 导致了一个侵袭性癌症家族虽然FUS已经成为主要的模型系统， 了解生物相分离，将FUS组件及其结构连接在一起的接触 在生理学和疾病中的作用是目前未知的，因为它们对传统技术是不可见的， 结构生物学然而，我们已经证明，我们可以可视化动态装配的FUS与 残差级分辨率。该项目将应用先进的核磁共振光谱，分子 模拟和FUS功能的细胞模型，以1）可视化介导LLPS的分子接触， 细胞相互作用，2）探测家族性FUS中发现的LC和RGG突变的分子和细胞影响， ALS，以及3）确定FUS与RNA和C-末端的复合物的功能相关的原子细节。 与RNA加工和转录相关的RNA聚合酶II的结构域（CTD）。FUS的这些研究 组装将提供关于用于抑制的未来药理学靶点的必要结构/功能信息。 ALS、FTD、白血病和肉瘤类型中的病理蛋白质关联。此外，由于FUS 只有一个许多基本的RNA结合蛋白含有聚集倾向的低复杂性结构域， 该项目的结果将作为理解整个蛋白质类的基础， 他们在疾病中的功能障碍。

Project Summary RNA-binding proteins are essential components of numerous large complexes that carry out fundamental processes including transcription, splicing, and DNA repair. Many RNA-binding proteins possess regions predicted to be disordered based on low-complexity sequence characteristics that are critical to normal RNA- processing functions, but also drive aberrant protein assembly in various neurodegenerative disease and cancers. The molecular interactions and functional roles of these disordered domains remain incompletely characterized, especially in the context of disease. Fused in Sarcoma (FUS) is one of twenty-nine human RNA-binding proteins that contains both an essential disordered low-complexity domain (LC) with unusually low charged residue composition and a high frequency of aromatic amino acids as well as several RGG motif regions. Despite disorder, these domains are thought to facilitate interactions in normal RNA metabolism by forming dynamic associations, thereby enabling tunable, reversible spatial clustering. Yet, excessive self- association between FUS disordered domains is believed to result in the formation of pathological neuronal inclusions in sub-types of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), irreversible neurodegenerative diseases that lack effective treatments. Moreover, fusion of the FUS disordered domains to several DNA-binding domains through chromosomal translocations results in uncontrolled gene expression leading to a family of aggressive cancers. Though FUS has emerged as the primary model system for understanding biological phase separation, the contacts holding together FUS assemblies and their structures in physiology and disease are currently unknown because they are invisible to traditional techniques in structural biology. However, we have demonstrated that we can visualize dynamic assemblies of FUS with residue-level resolution. This project will apply advanced nuclear magnetic resonance spectroscopy, molecular simulation, and cell models of FUS function to 1) visualize the molecular contacts that mediate LLPS and in- cell interactions, 2) probe the molecular and cellular impact of LC and RGG mutations of FUS found in familial ALS, and 3) determine the functionally relevant atomic details of FUS complexes with RNA and the C-terminal domain (CTD) of RNA polymerase II associated with RNA processing and transcription. These studies of FUS assembly will provide necessary structure/function information on future pharmacological targets for inhibiting pathological protein associations in types of ALS, FTD, leukemia, and sarcoma. Furthermore, because FUS is only one of many essential RNA-binding proteins containing aggregation-prone low complexity domains, the results of the project will serve as a foundation for understanding an entire class of proteins and for correcting their dysfunctions in disease.