Periodic Reporting for period 3 - PhaseAge (The chemistry and physics of RNP granules: how they form, age and cause disease)
Reporting period: 2019-05-01 to 2020-10-31
We recently proposed a new concept for the formation of RNP granules based on the principle of phase separation. In phase separation, an initially homogeneous solution of RBPs and RNAs separates into a dilute and a dense liquid phase, and the dense phase is highly enriched for RNAs and RBPs thus forming an RNP granule. We showed that with time the liquid-like state of RNP granules transitions into a solid-like, aberrant state that is associated with disease. In PhaseAge, we 1) investigate the molecular mechanisms of RNP granule formation, 2) study the molecular events that lead to aberrant RNPs, focusing on disease-associated mutations, changes in environmental conditions, post-translational modifications and molecules with fluidizing or solidifying effects, and 3) define the mechanisms of RNP granule quality control, which prevent aberrant phase transitions or reverse RNP granule aggregates to their normal state, thus rescuing a cell from an otherwise fatal condition.
We expect that our findings will give important insights into the molecular mechanisms of RNP granule formation and the molecular events that drive age-related changes that ultimately cause diseases such as ALS and FTD. We envision that our findings will lead to new therapeutic interventions that may significantly improve the prospects of patients afflicted with these diseases.
Using an in vitro reconstitution approach, we have recently succeeded for first time in rebuilding bona fide RNP granules in the test tube. We focused on stress granules that form when stressed cells shut down translation and release mRNA molecules from polysomes. Previous work had shown that stress granule formation is dependent on the RNA-binding protein G3BP. We show that under non-stressed conditions, the intrinsically disordered acidic tracts and positively charged arginine-rich regions of G3BP are sequestered by electrostatic intramolecular interactions to stabilize a compact, auto-inhibited state. Upon release from polysomes, unfolded mRNA molecules outcompete the auto-inhibitory intramolecular interaction, engendering a conformational transition that facilitates microclustering of G3BP through protein-RNA interactions. Subsequent physical crosslinking of G3BP microclusters drives RNA molecules into networked RNA/protein condensates. We show that G3BP condensates impede RNA entanglement and recruit additional client proteins such as FUS and TDP43, which promotes SG maturation. We propose that condensation by initiator molecules such as G3BP coupled to conformational rearrangements and heterotypic multivalent interactions may be a general principle underlying RNP granule formation.
We have also made extensive progress in understanding the protein-intrinsic molecular 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 showing that the measured saturation concentrations of phase separation are inversely proportional to the product of the numbers of arginine and tyrosine residues. These results provide us with an improved molecular understanding of disease-related mutations in RBPs and suggest that it will soon be possible to predict phase separation properties based on amino acid sequences.
We have further studied the mechanisms of how RBPs such as FUS phase separate to form liquid-like RNP condensates that then harden into less dynamic pathological structures. We find that phase separation of these RBPs 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 drive the transition into a solid-like state and thus the material properties of the condensates. We further demonstrate that the phase behavior of RBPs 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 RBPs cause aberrant phase transitions and disease. Additionally, we have 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 behavior 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. Any insult that leads to perturbations of this buffered could lead to disease. These new insights constitute a critical advance because they suggest pathways for how diseases such as ALS and FTD could arise.
Finally, we could provide evidence that proteins with weak folding mutations partition into reconstituted RNP granules and promote a liquid-to-solid transition, suggesting that aberrant physical interactions between liquid-like condensates and destabilizing protein folding polymorphisms can serve as nucleation sites for protein aggregation and the solidification of RNP granules. We obtained further evidence that the first line of defense against aberrant proteins conformations in RNP granules is formed by molecular chaperones. Indeed, we could show that diverse chaperone systems are recruited to SGs to maintain their liquid-like properties. We used purified components to reconstitute the quality control machinery of RNP granules in vitro. We found that the small heat shock protein (sHSP) HspB8 plays a central role in maintaining the liquid properties. A disease-associated mutation attenuates the ability of HspB8 to stop the molecular aging process. In contrast, the general sHSP HspB1 preferentially targets misfolded proteins that accumulate in the liquid droplet phase of SGs. We propose that aberrant phase transitions within liquid-like RNP granules lie at the heart of age-related diseases such as ALS and that the cellular chaperone machinery specifically targets and controls these processes.