Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

The liquid−liquid phase separation (LLPS) of proteins and RNA molecules has emerged in recent years as an important physicochemical process to explain the organization of membrane-less organelles in living cells and cellular functions and even some fatal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) due to the spontaneous condensation and growth of LLPS droplets. In general, the characterization of LLPS droplets has been performed by optical microscopy, where we need transparent substrates. By virtue of the liquid and wetting properties of LLPS droplets on a glass surface, there have been some technical protocols recommended to immobilize droplets on the surfaces. However, interactions between LLPS droplets and glass surfaces still remain unclear. Here, we investigated the surface diffusion of LLPS droplets on the glass surface to understand the interactions of droplets in a dynamic manner, and employed chemically modified glass surface with charges to investigate their Coulombic interaction with the surface. Using the single-particle tracking method, we first analyzed the diffusion of droplets on an untreated glass surface. Then, we compared the diffusion modes of LLPS droplets on each substrate and found that there were two major states of droplets on a solid surface: fix and diffusion mode for the LLPS droplet diffusion. While untreated glass showed a diffusion of droplets mainly, chemically modified glass with positive charges exhibited droplets fixed on the surface. It could arise from the Coulombic interaction between droplets and solid surface, where LLPS droplets have a negative ζ-potential. Our findings on the dynamics of LLPS at the solid/liquid interface could provide a novel insight to advance fundamental studies for understanding the LLPS formation.

Accretion and the resulting increase in local concentration is a widespread mechanism in biology to enhance biomolecular functions (for example, in liquid-liquid demixing phases). Such macromolecular aggregation phases (e.g., coacervates, amyloids) may also have played a role in the origin of life. Here, we report that a hydrophobic-cationic RNA binding peptide selected by phage display (P43: AKKVWIIMGGS) forms insoluble amyloid-containing aggregates, which reversibly accrete RNA on their surfaces in an RNA-length and Mg2+-concentration dependent manner. The aggregates formed by P43 or its sequence-simplified version (K2V6: KKVVVVVV) inhibited RNA polymerase ribozyme (RPR) activity at 25 mM MgCl2, while enhancing it significantly at 400 mM MgCl2. Our work shows that such hydrophobic-cationic peptide aggregates can reversibly concentrate RNA and enhance the RPR activity, and suggests that they could have aided the emergence and evolution of longer and functional RNAs in the fluctuating environments of the prebiotic earth.