Molecular Insights into the Role of RNA Structure in the Phase Separated Nucleolus

Abstract

Ribosome biogenesis is a critical cellular process and its dysregulation is associated with altered cellular behavior, as well as morbidity and death in humans. Biogenesis and assembly of these complex molecular machines occurs in the nucleolus, a large compartmentalized membrane-less organelle formed through liquid-liquid phase separation (LLPS). The protein Nucleophosmin (NPM1) is a major constituent of the outer, granular component (GC) region of the nucleolus wherein it phase separates with ribosomal proteins (r-proteins) and ribosomal RNA (rRNA). Despite the biological importance of NPM1/rRNA interactions in ribosome assembly, the physiochemical basis of these interactions is poorly understood. Preliminary data demonstrates that rRNA structure influences rRNA/NPM1 LLPS, suggesting a structure-based regulatory mechanism. Mg2+ addition significantly enhances and alters the observed fluid properties of NPM1/rRNA droplets. Chemical (SHAPE-MaP) and biophysical studies correlate observed enhanced LLPS with an increase in nascent RNA structure. Seminal studies in the 1970s by Nomura and others (Nomura, 1973) showed that during assembly, association of r-proteins act to stabilize and/or reshape rRNA into conformations(s) conducive to later r-protein association. Data presented herein further demonstrate binding of r-proteins to rRNA abrogates rRNA/NPM1 phase separation,suggesting a mechanism wherein sequential association of r-proteins leads to the occlusion of NPM1 binding sites and allows for release of assembled pre-ribosomal particles into the nucleoplasm. Broadly, our studies provide novel insight into how RNA structure can actively regulate phase separation in the nucleolus and suggest how leukemia associated NPM1 mutations may alter nucleolar LLPS, deregulate ribosome biogenesis, and promote oncogenesis.