Visualizing Structural Details of Disordered Domain Phase Separation Associated with ALS and Cancers

Abstract

Phase separation of RNA-binding proteins via multivalent interactions between aromatic/polar-rich disordered domains contributes to the formation of functional cytoplasmic granules and nuclear puncta. These domains have also been identified as the nucleators of neuronal inclusions in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We use atomic resolution nuclear magnetic resonance spectroscopy approaches for visualizing low complexity domain structure and interactions along the pathway from monomer, to liquid-liquid phase separated state, to static aggregates and hydrogels. We show that the low complexity domain of RNA-binding protein Fused in Sarcoma (FUS LC) remains disordered even within liquid phase separated states and recruits unphosphorylated RNA-polymerase II C-terminal domain into the liquid phase separated state, adding a potential explanation for FUS LC transcriptional activation in cancer. Importantly, phase separation is reversible and is modulated by ionic strength and interaction with RNA, distinguishing these assemblies from static inclusions. In contrast, we show that liquid-liquid phase separation of TDP-43 is mediated in part by α-helical assembly and extension. Some ALS-associated mutations disrupt helix-helix interaction inhibiting liquid-liquid phase separation while leading to enhanced aggregation.