Atomic details of protein/RNA liquid-liquid phase separation by experiment and simulation

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, which have been shown to behave as liquids -- flowing, fusing, and returning to spherical shape -- within live cells. These domains have also been identified as the nucleators of cytoplasmic inclusions associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We use atomic resolution nuclear magnetic resonance spectroscopy approaches to visualize 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 domains of RNA-binding protein Fused in Sarcoma (FUS LC, associated with ALS and FTD) and hnRNP A2 (associated with ALS and IBM) remain disordered even within liquid phase separated puncta (Burke et al. Molecular Cell, 2015). FUS LC also 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 interaction with RNA, distinguishing these assemblies from static inclusions that can arise from missense mutations in the LC regions. In contrast, we show that liquid-liquid phase separation of TDP-43 is mediated in part by structured α-helical assembly and extension (Conicella et al. Structure, 2016). Some ALS-associated mutations disrupt helix-helix interaction inhibiting liquid-liquid phase separation while leading to enhanced aggregation. Our current work aims to evaluate the potential of post translational modification to alter assembly and hence disrupt pathological interactions of these disordered domains (Monahan and Ryan et al. EMBO J 2017).