Cell-free Formation of RNA Granules: Low Complexity Sequence Domains Form Dynamic Fibers within Hydrogels

Abstract

Eukaryotic cells contain assemblies of RNAs and proteins termed RNA granules. Many proteins within these bodies contain KH or RRM RNA-binding domains as well as low complexity (LC) sequences of unknown function. We discovered that exposure of cell or tissue lysates to a biotinylated isoxazole (b-isox) chemical precipitated hundreds of RNA-binding proteins with significant overlap to the constituents of RNA granules. The LC sequences within these proteins are both necessary and sufficient for b-isox-mediated aggregation, and these domains can undergo a concentration-dependent phase transition to a hydrogel-like state in the absence of the chemical. X-ray diffraction and EM studies revealed the hydrogels to be composed of uniformly polymerized amyloid-like fibers. Unlike pathogenic fibers, the LC sequence-based polymers described here are dynamic and accommodate heterotypic polymerization. These observations offer a framework for understanding the function of LC sequences as well as an organizing principle for cellular structures that are not membrane bound.

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…The growth rate and extent of droplet formation depended on the degree of phosphorylation, whereby the introduced negative charges (as in tau441E17) could only account for some of this effect; it seems likely that the addition of phosphate groups at certain positions is relevant for the conformational changes that can lead to LLPS of tau. The lack of tau droplet coalescence suggests a fast increase in the viscoelasticity of phospho‐tau droplets preventing droplet fusion (Abbott, 1977; Gu et al , 2011); this is characteristic for the “maturation” of liquids into hydrogels (Kato et al , 2012). The subsequent deformation of p‐tau441 droplets and the formation of tau aggregates in the droplets are strong indicators for the further transition of the tau droplets from a gel‐like into an aggregate state of lower energy; a similar maturation has been described for FUS (Patel et al , 2015).…”

“…To evaluate the transition from a liquid to a gel‐like state, which is characterized by an increase in viscoelasticity and, hence, a decrease in molecular diffusion rates in the gel state (Kato et al , 2012), we performed FRAP microscopy of freshly prepared p‐tau441 droplets that contained 10% of fluorescently labeled protein of the same kind (p‐tau441‐dl488). When bleaching the entire droplets (Fig 4A) at different times after droplet initiation (addition of 10% PEG), we found a rapid decrease of recovery in the first 15 min and virtually no recovery after 30 min.…”

Phosphorylation of the FUS low‐complexity domain disrupts phase separation, aggregation, and toxicity

“…Recent work strongly points to the ability of these domains to form functional interactions with specific sets of proteins bearing similar low‐complexity (or prion‐like) domains (Kato et al , 2012; Molliex et al , 2015). Currently, many questions remain about the molecular nature of these interactions inside RNP granules—that is, where each specific membraneless organelle falls on a continuum of molecular architectures (Aguzzi & Altmeyer, 2016) ranging from structured cross‐β amyloids to disordered polymer chains (Patel et al , 2015; Xiang et al , 2015; Boke et al , 2016).…”

“…Further efforts are needed to determine how phase separation is governed in cells where a multitude of components make up physiological granules. FUS‐containing membraneless organelles are not homogeneous structures composed of one protein and one RNA (Kato et al , 2012; Jain et al , 2016). The addition of other granule components and molecular chaperones in vivo may decrease the dominant role of charge–charge repulsion in the effect on phase separation observed in vitro .…”

Prions, amyloids, and RNA: Pieces of a puzzle

“…[106][107][108][109] These granules contain non-translating mRNAs and are involved in the control of all stages of mRNA turnover, including storage and degradation. The structure of these granules is highly dynamic, as their protein and RNA composition undergoes significant changes depending on the particular needs of the cell.…”