Negative regulation of a ribonucleoprotein condensate driven by dilute phase oligomerization

Abstract

Ribonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms ribonucleoprotein condensates with different RNA molecules, and these condensates are implicated in key processes such as cell-cycle control and generating cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region (IDR) and a tandem RNA recognition module. Here, we demonstrate that a 21-residue stretch within the Q-rich IDR has a weak intrinsic preference for forming alpha-helical conformations. Through mutagenesis, we find that increased alpha helicity enhances oligomerization in the dilute phase. One consequence of enhanced oligomerization is a dilution of Whi3 in the dense phase. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, the formation of dilute phase oligomers, driven by a specific sequence motif and potential synergies with the rest of the IDR, opposes incorporation of the Whi3 protein into the dense phase, thereby altering the dense phase stoichiometry of protein to RNA. Our findings, which stand in contrast to other systems where oligomerization has been shown to enhance the drive for phase separation, point to a novel mechanism that might be operative for influencing compositions of condensates. Our work also points to routes for designing synthetic ribonucleoprotein condensates whereby modulation of protein oligomerization via homotypic interactions can impact dense phase concentrations, stoichiometries, and material properties. SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and are known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces for biomolecular condensate formation, relatively little is known about forces that oppose assembly. Here, using a fungal RNP protein, Whi3, we show that a portion of its intrinsically disordered, glutamine-rich region modulates phase separation by forming transient alpha helical structures that promote the assembly of dilute phase oligomers. These oligomers detour Whi3 proteins from condensates, thereby impacting the driving forces for phase separation, the protein-to-RNA ratio in condensates, and the material properties of condensates. Our findings show how nanoscale conformational and oligomerization equilibria can influence mesoscale phase equilibria.