Sedimentation Assays to Assess the Impact of Posttranslational Modifications on Phase Separation of RNA-Binding Proteins In Vitro and In Cells

Abstract

In the last years, RNA-binding proteins (RBPs) have been highlighted for their capacity to undergo liquid-liquid phase separation (LLPS). Aberrant phase transitions of RBPs from a liquid to a solid state are believed to underlie the formation of pathological RBP aggregates in several neurodegenerative diseases. Both in the physiological and the disease state, RBPs are often decorated with diverse posttranslational modifications (PTMs) that can influence the phase separation behavior, the physiological function, and the pathological behavior of the RBP. Here we describe two simple methods, sedimentation assays in vitro and in cells, that allow the analysis of RBP solubility as a measure of RBP phase separation in the absence or presence of a certain PTM.