Formation of versus Recruitment to RNA-Rich Condensates: Controlling Effects Exerted by Peptide Side Chain Identity

Abstract

Liquid−liquid phase separation (LLPS), the spontaneous formation of contiguous liquid phases with distinct compositions, has been long known in chemical systems and more recently recognized as a ubiquitous feature of cell biology. We describe a system involving biologically relevant components, synthetic peptides, and total yeast RNA, that has enabled us to explore factors that underlie phase separation. Coulombic complementarity between a cationic peptide and anionic RNA is necessary but not sufficient for formation of a condensed phase in our system. In addition to a net positive charge, the peptide must present the proper type of cationic moiety. Guanidinium groups, as found in the Arg side chain, support phase separation, but ammonium groups, as found in the Lys side chain, or dimethylguanidinium groups, as found in post-translationally modified Arg side chains, do not support phase separation in our system. However, the cationic groups that do not support phase separation via interaction with RNA can nevertheless enable recruitment to a condensed phase, which reveals that the network of forces governing condensed phase formation can differ from the network of forces governing recruitment to such a phase. We introduce a new method for measuring the concentrations of components in condensed phases based on fluorine-containing additives and 19 F NMR.

Determining the Physico‐Chemical Composition of Biomolecular Condensates from Spatially‐Resolved NMR

“…Multi‐component quantification can be performed in a temperature‐dependent manner in real time providing insight into the kinetics of condensation processes. The improved WET‐DPFGSE NMR experiment thus complements and extends previous NMR approaches that relied on the use of fluorinated molecules for quantification of protein or small molecule concentrations in phase‐separated systems [13, 17] . In addition, Raman microscopy can be useful to quantify protein concentrations, on the basis of the signals emerging from the different amounts of water present in the condensed and dilute phase [18] …”

“…The improved WET-DPFGSE NMR experiment thus complements and extends previous NMR approaches that relied on the use of fluorinated molecules for quantification of protein or small molecule concentrations in phase-separated systems. [13,17] In addition, Raman microscopy can be useful to quantify protein concentrations, on the basis of the signals emerging from the different amounts of water present in the condensed and dilute phase. [18] The improved spatially-resolved NMR is applicable to effectively any IDP as long as its NMR resonances are observable.…”

Development of Highly Fluorogenic Styrene Probes for Visualizing RNA in Live Cells

“…Hence, the use of fluorescently labeled condensate components can enable their detection in vitro and in cells. Previous studies have utilized fluorescently labeled RNA, , peptides, and proteins − to visualize RNA coacervates, a type of droplet formed by LLPS. However, these strategies require modifying RNA and engineering proteins with exogenous fluorophores such as fluorescein and GFP.…”

Visualizing formation and dynamics of a three-dimensional sponge-like network of a coacervate in real time

“…A coacervate is a condensed fluid that forms by the liquid-liquid phase separation (LLPS) of small organic molecules, polypeptides, pairs of oppositely charged polyelectrolytes, or biomacromolecules in aqueous solution. [1][2][3][4][5][6][7][8] Coacervates are highly dynamic. They exhibit a variety of unique behaviors, including coalescence into larger spherical assemblies upon contact with one another; facilitated chemical reactions upon internal molecular sequestration; and metastability by transforming into solid-like amyloid fibrils.…”