Molecular insights into the homotypic and heterotypic interactions that promote liquid-liquid phase separation of the RNA-binding protein Fused In SarcomadissertationsMurthy, Anastasia CcreatorFawzi, NicolasAdvisorHart, AnneReaderJogl, GerwaldReaderLisi, GeorgeReaderMowry, KimberlyReaderBosco, DarylReaderBrown University. Department of Molecular Biology, Cell Biology and Biochemistrysponsor2020xv, 232 p.born digitalThesis (Ph. D.)--Brown University, 2020thesesSubcellular organization is important for maintaining cellular homeostasis within eukaryotic cells. In addition to membrane-bound organelles, membraneless compartmentalization of the cell occurs via liquid-liquid phase separation (LLPS) of proteins and nucleic acids. The underlying molecular interactions that underpin biomolecular LLPS have been of increased interest due to the importance of membraneless organelles (MLOs) in facilitating various biological processes and the disease-association of several of the proteins that mediate LLPS. Proteins that are able to undergo LLPS often contain intrinsically disordered regions. Solution-state nuclear magnetic resonance (NMR) spectroscopy has emerged as a leading structural technique to characterize protein LLPS due to the variety and specificity of information that can be obtained about intrinsically disordered sequences. Fused in Sarcoma (FUS) is a ribonucleoprotein which functions in multiple aspects of RNA metabolism including transcription. FUS is able to undergo LLPS; however, the molecular details about the homotypic and heterotypic protein-protein interactions that contributed to FUS LLPS and incorporation into MLOs is unknown. In Chapter Two, I used solution-state NMR spectroscopy to directly characterize the secondary structure within the condensed phase of FUS SYGQ LC. I found that FUS SYGQ LC retains conformational heterogeneity within the condensed phase, allowing for multivalent interactions involving all major residue types. In Chapter Three, I extended this analysis to understand the sequence motifs that contribute to LLPS as opposed to fibrillar aggregation. Using bioinformatics and mutational analysis, I identify GYGQ motifs as important for liquid-like assembly. Finally, in Chapter Four, I demonstrate that the RGG domains of FUS contribute to LLPS in concert with the SYGQ LC and interact with RNA polymerase II. My work provides a unified mechanism for FUS LLPS involving both the N-terminal SYGQ-rich low complexity domain and RGG domains and supports the hypothesis that conformational heterogeneity and multivalent interactions are important for LLPS systems in general. Together, this thesis establishes the mechanistic basis of FUS LLPS and lays the foundation for insights into the role of FUS in RNP granules.Nuclear magnetic resonance spectroscopyliquid-liquid phase separationRNP granulesFused in SarcomaStructural biologyProtein assemblyEnglishRPB20220912