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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - PhaseAge (The chemistry and physics of RNP granules: how they form, age and cause disease)

Teaser

Summary

Liquid-liquid phase separation can create functionally distinct reaction compartments consisting of proteins and RNAs called ribonucleoprotein (RNP) granules, which have major roles in cellular organization and physiology. Prion-like RNA-binding proteins such as Fused in Sarcoma (FUS) are the key players that mediate the process of RNP granule formation by phase separation in cells. Recent data suggests that aberrant phase transitions of these proteins from a liquid to a solid-like state may be closely tied to the pathogenesis associated with diseases such as amyotrophic lateral sclerosis (ALS). Thus, elucidating how physiological phase separation gives rise to aberrant phase transitions and dysfunctional RNP granules will be key to understand these neurodegenerative diseases.

We investigated how RNA-binding protein such as FUS phase separate to form liquid-like RNP condensates that harden into less dynamic pathological structures. We find that phase separation is primarily governed by multivalent interactions among amino acid motifs that we call stickers. We show that these stickers are connected by flexible spacer sequences that govern the material properties of the condensates. We further demonstrate that the phase behaviour of FUS in vitro critically depends on the RNA concentration: low RNA/protein ratios promote phase separation into liquid condensates, whereas high ratios prevent condensate formation. Moreover, reduction of RNA levels in cells causes excessive phase separation and the formation of pathological solid-like structures. Based on these data, we propose that phase separation is driven by a protein-intrinsic molecular grammar and that changes in RNA levels or RNA binding abilities of RNA-binding proteins cause aberrant phase transitions and disease.

Work performed

We made extensive progress in understanding the rules underlying the formation of RNP granules. We used mutagenesis to identify a sequence-encoded molecular grammar underlying the driving forces of phase separation of FUS and related prion-like proteins and we tested aspects of this grammar in cells. We found that phase separation is primarily governed by multivalent interactions among tyrosine residues from prion-like domains and arginine residues from RNA-binding domains, which are modulated by negatively charged residues. Glycine residues enhanced the fluidity, whereas glutamine and serine residues promoted hardening. Based on this insight, we developed a model to show that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results suggest it is possible to predict phase separation properties based on amino acid sequences.
We also gained important insight into how RNP granule formation is regulated. Prion-like RNA binding proteins (RBPs) such as TDP43 and FUS are normally soluble in the nucleus but form solid pathological aggregates when mislocalized to the cytoplasm. We could show that RNA critically regulates the phase behaviour of prion-like RBPs. Low RNA/protein ratios promote phase separation into liquid droplets, whereas high ratios prevent droplet formation in vitro. Based on these findings, we propose that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble. This suggests that the nucleus is a buffered system in which high RNA concentrations keep RBPs soluble.

Final results

We have gained important insights into the rules governing phase separation properties and dynamics. Our results suggest that we should soon be abble to predict phase separation properties based on amino acid sequence. We also expect that we will soon be able to control phase separation with targeted genetic and chemical approaches.