Condensation of ribosomal proteins in ribosome biogenesis and neurological ribosomopathy

核糖体生物发生和神经性核糖体病中核糖体蛋白的缩合

• 批准号: 506373047
• 负责人: Dr. Matthew Kraushar, Ph.D.
• 金额: --
• 依托单位: Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE)
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/506373047?language=en
• 关键词: Condensation; ribosomal; proteins; ribosome; biogenesis

Liquid-liquid phase separation (LLPS) is a highly efficient mechanism to concentrate molecules for biological reactions. The nucleolus is a liquid condensate with the principal function of assembling ribosome subunits, a membrane-less compartment for transcribing rRNA and supporting ribosomal protein (RP) interactions. RPs are the most abundant proteins in the nucleolus. However, whether RPs individually or collectively undergo liquid-liquid phase separation to form condensates miscible with the nucleolus, concentrating around rRNA within, is unknown. This SPP2191 Proposal aims to dissect the LLPS character of RPs and their interactions within the nucleolus, towards the functional assembly of ribosomal subunits. Our approach will leveraging a unique combination ribosome-specific and LLPS-specific expertise, utilizing methods in biochemisty, biophysics, cells biology, and high-resolution structural biology.Both the nucleolus and the ribosome itself have undergone substantial evolutionary advancements. The surrounding nucleus is present in eukaryotes, and absent in prokaryotes; however, bacteria contain a “nucleoid region” where ribosomes are assembled. 79 RPs are required to assemble the human ribosome, and 55 RPs in E. coli. Whether the LLPS characteristic of the eukaryotic nucleolus is related to the evolution of ribosome assembly and structure is unknown. Our preliminary bioinformatic data predict that the evolution of human RPs greatly expands their content of intrinsically disordered regions (IDRs), which are drivers of LLPS for many other proteins. We leverage our unique experience with fully in vitro reconstituted ribosome subunit assembly to find that the ribosomal protein pool forms condensates, which are dissolved with the addition of rRNA that triggers an entropically-driven sequential assembly reaction. This project will employ this in vitro ribosome assembly system, and monitor LLPS of RPs in E. coli and human cells by fluorescence/FLIM microscopy. Ribosome biochemistry and cryo-electron microscopy will measure the functional output of RP-IDR-driven condensation in ribosome assembly.Finally, this project will study the role of RP condensation in a neurodegenerative “ribosomopathy”. Alzheimer’s disease is known to demonstrate abnormal protein synthesis by the ribosome, but the mechanism is unknown. Tau is a pathogenic protein causing Alzheimer’s disease by abnormal aggregation, and has been shown by our team to undergo LLPS. Our preliminary data indicate Tau interacts with many RPs, and thus we aim to dissect how the myriad of Tau-RP interactions may be initiated in mixed condensates, as a mechanism for abnormal protein synthesis by the ribosome in Alzheimer’s disease.Taken together, our SPP2191 Proposal will spearhead an understanding of the molecular landscape of ribosome assembly in the evolution of LLPS condensates and the ribosome itself.

液-液相分离（LLPS）是一种高效的浓缩生物反应分子的机制。核仁是一种液体凝聚物，其主要功能是组装核糖体亚单位，是一个用于转录rRNA和支持核糖体蛋白（RP）相互作用的无膜隔室。RP是核仁中最丰富的蛋白质。然而，RP是否单独或共同经历液-液相分离，形成与核仁混溶的浓缩物，浓缩在rRNA周围，是未知的。这个SPP 2191提案旨在剖析RPs的LLPS特征及其在核仁内的相互作用，以实现核糖体亚基的功能组装。我们的方法将利用核糖体特异性和LLPS特异性的独特组合，利用生物化学，生物物理学，细胞生物学和高分辨率结构生物学的方法。核仁和核糖体本身都经历了实质性的进化进步。核周围存在于真核生物中，而在原核生物中不存在;然而，细菌含有核糖体组装的“类核区域”。人核糖体的组装需要79个RP，而E.杆菌真核细胞核仁的LLPS特征是否与核糖体组装和结构的进化有关尚不清楚。我们的初步生物信息学数据预测，人类RP的进化大大扩展了其内在无序区（IDR）的内容，这是许多其他蛋白质的LLPS的驱动程序。我们利用我们在完全体外重组核糖体亚基组装方面的独特经验，发现核糖体蛋白池形成缩合物，这些缩合物在加入rRNA后溶解，从而引发熵驱动的顺序组装反应。本项目将利用该体外核糖体组装系统，对大肠杆菌中核糖体的LLPS进行监测。大肠杆菌和人细胞的荧光/FLIM显微镜。核糖体生物化学和冷冻电子显微镜将测量核糖体组装中RP-IDR驱动的缩合的功能输出。最后，本项目将研究RP缩合在神经退行性“核糖体病”中的作用。阿尔茨海默氏病是已知的，证明异常蛋白质合成的核糖体，但机制是未知的。Tau是一种通过异常聚集引起阿尔茨海默病的致病蛋白，并且已经被我们的团队证明经历LLPS。我们的初步数据表明Tau与许多RP相互作用，因此我们的目标是剖析如何无数的Tau-RP相互作用可能在混合缩合物中启动，作为阿尔茨海默病中核糖体异常蛋白质合成的机制，我们的SPP 2191提案将率先了解LLPS缩合物和核糖体本身进化中核糖体组装的分子景观。