Condensation of ribosomal proteins in ribosome biogenesis and neurological ribosomopathy

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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.