Biochemical characteristics of spacer amino acid determine phase-separating behavior and induction of nucleolar stress by arginine-rich dipeptide repeat proteins

Arginine-rich dipeptide repeat proteins (R-DPRs), produced from hexanucleotide repeat expansions via repeat-associated non-AUG translation, are linked to neurodegenerative disorders. While novel, potentially pathogenic R-DPRs have been identified, their intracellular signaling mechanisms remain unclear. To explore their biological roles, we examined R-DPR liquid–liquid phase separation behavior in vitro and their impact on nucleolar function. We found that the spacer hydrophobicity is a key determinant of R-DPR phase separation. Specifically, R-DPRs with hydrophobic spacers formed aggregates with RNA and recombinant nucleophosmin in vitro, inducing nucleolar stress and impairing ribosomal RNA synthesis in cells. These findings provide insights into the mechanisms by which R-DPRs exert nucleolar stress, advancing our understanding of their pathological roles.