Assay to Study the Phase-transition Behavior of Edc3, a Conserved Processing Body (P-body) Marker Protein

Abstract

RNA granules are conserved, non-membranous, biphasic structures predominantly composed of RNA and RNA-binding proteins. RNA granules often assemble as a result of cellular responses to a variety of stresses, including infection. Two types of RNA granules are best characterized: stress granules (SGs) and processing bodies (P-bodies). The mechanism of RNA granule assembly and disassembly is still understudied because of its complex composition and dynamic behavior. The assembly of RNA granules is driven by a process known as phase separation of granule components. Edc3 is a conserved decapping activator and an essential P-body component in Saccharomyces cerevisiae. Phase separation of P-body proteins has been poorly explored. This protocol will enable the visualization of the phase transition behavior of Edc3, since it is tagged to mCherry. It further describes using small molecules and other proteins to study P-body dynamics. In addition to the assembly of Edc3, this assay also lays the foundation to study disassembly of phase-separated assemblies in vitro, which was not explored earlier. We have devised the assay to describe the use of one such protein that acts as a disassembly factor. Overall, this protocol is simple to perform and can potentially be combined with analyzing these assemblies using other approaches.Graphical abstract: