Condensation of ribosomal proteins in ribosome biogenesis and neurological ribosomopathy

液-液相分离是一种高效的生物反应分子浓缩机制。核仁是一种液体凝结物，其主要功能是组装核糖体亚基，这是一种用于转录rRNA和支持核糖体蛋白（RP）相互作用的无膜室。RPs是核仁中含量最多的蛋白质。然而，RPs是单独还是集体进行液-液相分离，形成与核仁混溶的凝聚物，集中在其中的rRNA周围，尚不清楚。SPP2191提案旨在剖析RPs的LLPS特征及其在核核内的相互作用，以实现核糖体亚基的功能组装。我们的方法将利用独特的核糖体特异性和llps特异性专业知识，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。周围核存在于真核生物中，不存在于原核生物中；然而，细菌含有一个核糖体组装的“类核区”。人类核糖体需要79个RPs，大肠杆菌需要55个RPs。真核生物核核的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RPs的进化极大地扩展了其内在无序区（IDRs）的含量，这是许多其他蛋白质LLPS的驱动因素。我们利用我们在体外完全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凝聚体，这些凝聚体在加入rRNA时溶解，从而触发熵驱动的顺序组装反应。本项目将采用该体外核糖体组装系统，通过荧光/FLIM显微镜监测大肠杆菌和人细胞中RPs的LLPS。核糖体生物化学和低温电镜将测量核糖体组装中rp - idr驱动的冷凝的功能输出。最后，本项目将研究RP缩聚在神经退行性“核糖体病”中的作用。已知阿尔茨海默病表现为核糖体异常蛋白质合成，但其机制尚不清楚。Tau是一种通过异常聚集导致阿尔茨海默病的致病蛋白，我们的团队已经证明Tau会经历LLPS。我们的初步数据表明Tau与许多rp相互作用，因此我们的目标是解剖Tau- rp相互作用如何在混合冷凝物中启动，作为阿尔茨海默病中核糖体异常蛋白质合成的机制。综上所述，我们的SPP2191提案将率先理解LLPS凝聚物和核糖体本身进化过程中核糖体组装的分子景观。

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.