NEPRO forms fibrous scaffolds in the nucleolus to maintain nucleolar integrity and orchestrate ribosome biogenesis

The nucleolus is a dynamic, membrane-less organelle whose sub-compartmentalization regulates ribosome biogenesis, RNA processing, and stress signaling. While Nucleolus and Neural Progenitor protein (NEPRO) has been classified as a nucleolar protein involved in ribosomopathy, its intra-nucleolar organization, structural features, and functional contributions have remained less understood. Here, we show that NEPRO localizes predominantly to the edges of the nucleolar boundary, forming distinct pools that do not localize to the canonical nucleolar subdomains. NEPRO assembles into elongated fibers in nucleolus. Recombinant NEPRO self-polymerizes into fibrils under mildly acidic conditions, and these fibers also contour the periphery of the dense fibrillar component of nucleolus. NEPRO depletion disrupted nucleolar integrity. NEPRO harbors an arginine-rich nucleolar localization signal (NoLS: 442-460 amino acid region), and interaction of the NoLS of NEPRO with GNL3's acidic C-terminal region nucleates and concentrates NEPRO, lowering its fibrillation threshold and promoting pH-dependent fibrillar assembly in nucleolus. Affinity pulldown of NEPRO's N-terminal coiled-coil domain identified nucleolar interacting proteins enriched in ribosomal and rRNA-processing factors. NEPRO knockdown impaired 40S/60S/80S ribosome assembly, induced G₀/G₁ arrest, and cell senescence. Collectively, our data define NEPRO as a structural scaffold protein that orchestrates nucleolar structural organization and ribosome biogenesis through formation of fibrous structures, with its dysfunction leading to nucleolar stress and cell-cycle arrest.