Phase separation via protein-protein and protein-RNA networks coordinates ribosome assembly in the nucleolus

The nucleolus, the largest membraneless organelle in the nucleus, functions as the site for ribosome biogenesis. While long known for its complex and dynamic structure, our mechanistic understanding of nucleolar organization has advanced dramatically in the past 15 years. The process of phase separation (PS) facilitates the compartmentalization of ribosomal components with assembly factors in the nucleolus, underlying complex ribosome biogenesis processes. Multicomponent PS creates multiple nucleolar sub-compartments that function from inside out as a ribosome assembly line. In this review, we discuss the molecular basis of nucleolar organization, including how different types of protein-protein and protein-RNA interactions create the multilayered architecture that enables ribosome biogenesis. Key proteins including nucleolin, fibrillarin, and nucleophosmin mediate nucleolar compartmentalization through their unique structural features and multivalent interactions. The processes of ribosomal RNA (rRNA) transcription, modification and splicing, and folding are spatially and temporally segregated within different regions of the nucleolus. rRNA matures and changes form along this processing continuum, continually altering its interactions with proteins, creating multiple separate liquid phases that establish sub-compartments. We highlight how both folded domains and intrinsically disordered regions (IDRs) in nucleolar proteins contribute to multivalent interactions underlying PS and nucleolar compartmentalization. We also discuss how perturbation of nucleolar PS alters nucleolar structure, dynamics, and function and contributes to a range of pathological conditions.