Ribosomal protein uL30 undergoes phase separation with nucleophosmin and regulates nucleolar formation in the absence of RNA.

The nucleolus is a membrane-less structure that exists in the nucleus of cells and plays a crucial role in ribosome biogenesis. It is known to be formed through liquid-liquid phase separation (LLPS) caused by the interaction of various nucleolar proteins and nucleic acids. Recently, many studies on LLPS with nucleolar proteins in the presence of RNA showed the importance of electrostatic interactions and cation-pi interactions among RNA and intrinsically disordered regions of proteins. However, it is reported that the initiation of nucleolar formation is RNA polymerase I-independent. The mechanism of nucleolar formation in the early stage remains obscure. In this study, we showed for the first time that the ribosomal protein uL30 and a major nucleolar protein, nucleophosmin (NPM) formed liquid droplets in vitro in the absence of RNA. The liquid droplet formation with uL30 and NPM may be derived from the interaction between the basic regions of uL30 and acidic regions of the oligomeric NPM. The knockdown of uL30 in cells significantly reduced the number of nucleoli, while it did not alter the protein level of NPM. The results showed that LLPS and nucleolar formation were affected by changes in uL30 levels. Our results suggest that the protein-protein interaction between nucleolar proteins may play an important role in nucleolar formation in the early stages when the rRNA content is very low.