Description
- Abstract:
- Liquid-liquid phase separation (LLPS) is a prominent organizer of cellular biochemistry. Dozens of liquid-like condensates are found throughout the nucleus and the cytoplasm and the vast majority of them are enriched in RNA-binding proteins; the maestros of gene expression. Fused in Sarcoma (FUS) is an RNA-binding protein that phase separates via its low-complexity (LC) and arginine- glycine-glycine (RGG) domains. FUS localizes in liquid-like assemblies like stress granules and DNA- repair foci when the cell needs to combat stress and DNA damage respectively. However, mutations and/or abnormal levels of post-translational modifications (PTMs) disorient physiologically relevant interactions of FUS with RNA and other binding partners leading to a number of neurodegenerative diseases and cancers with unmet therapeutic needs. Combining computer simulations with solution- state nuclear magnetic resonance (NMR) has emerged as an unparalleled method to obtain atomic- level details of liquid-like structures. In this thesis, the main focus is towards a deeper understanding of the biophysical rules governing the interplay of FUS with PTMs, RNA and condensate-modifying molecules. In Chapter 2, I use coarse-grained (CG) modeling to show how measuring the effect of PTMs like phosphorylation and acetylation on the single-chain properties of various systems including FUS is predictive of their propensity to phase separate. In Chapter 3, I decipher the behavior of SARS-CoV-2 nucleocapsid protein which phase separates with RNA as I was equipped to face this challenge during the course of my doctoral studies in LLPS. In Chapter 4, I use NMR and biochemical assays to probe how condensate-modifying molecules alter FUS structure, phase behavior and interactions with RNA. In Chapter 5, I demonstrate that arginine-specific contacts are necessary for FUS RGG-RNA interactions and methylation alters heterotypic interactions of FUS RGG with RNA and the LC domain. Collectively, my work provides a detailed molecular view into the effect of PTMs, RNA and drug-like molecules on RNA-binding protein assembly. This knowledge can be harnessed to design in-vitro drug-design studies that screen the effect of drug molecules against FUS LLPS and aggregation.
- Notes:
- Thesis (Ph. D.)--Brown University, 2022
Citation
Perdikari, Theodora Myrto,
"Biophysical studies of phase separation: RNA-binding proteins at the nexus of neurodegeneration, cancer and viral infection"
(2022).
Biomedical Engineering Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/7fnh-8877
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Biomedical Engineering Theses and Dissertations
Theses and Dissertations for the Biomedical Engineering department....