Dilute phase oligomerization can oppose phase separation and modulate material properties of a ribonucleoprotein condensate

Abstract

Ribonucleoprotein bodies are exemplars of membraneless biomolecular condensates that can form via spontaneous or driven phase transitions. The fungal protein Whi3 forms compositionally distinct ribonucleoprotein condensates that are implicated in key processes such as cell-cycle control and cell polarity. Whi3 has a modular architecture that includes a Q-rich intrinsically disordered region and a tandem RNA recognition module. Here, we uncover localized order-to-disorder transitions within a 21-residue stretch of the Q-rich region. This region, which can form alpha-helical conformations, is shown to modulate protein density within Whi3-RNA condensates by driving dilute phase oligomerization. Specifically, enhancing helicity within this region enhances oligomerization in the dilute phase. This weakens the associations among disordered Q-rich regions thereby diluting the concentration of Whi3 in condensates. The opposite behavior is observed when helicity within the 21-residue stretch of the Q-rich region is abrogated. Thus, dilute phase oligomers, driven by a specific sequence motif, lead to negative regulation of the stoichiometry of protein versus RNA in the dense phase. Our findings stand in contrast to other systems where oligomerization is known to enhance the drive for phase separation. Our results highlight distinctive regulatory effects over phase behavior due to local order-to-disorder transitions within intrinsically disordered regions. This provides a way to leverage molecular scale conformational preferences and coupled intermolecular associations to regulate mesoscale phase behavior and material properties of condensates.SignificanceA large sub-class of biomolecular condensates are linked to RNA regulation and known as ribonucleoprotein (RNP) bodies. While extensive work has identified driving forces of biomolecular condensates, relatively little is known about negative regulation of assembly. Here, using a fungal RNP component, Whi3, we show that its intrinsically-disordered, Q-rich region exerts regulatory control over condensate formation through a cryptic helical region that enables the formation 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 equilibria can enable control over micron-scale phase equilibria.

Structure and position-specific interactions of prion-like domains in transcription factor Efg1 phase separation

“…The self-interaction mediated by helical structures in Efg1 PrLD aligns with similar observations in proteins such as TDP-43 CTD (19,20), although we do not find evidence for helical enhancement upon self-assembly as observed for TDP-43 (19). Though the effect on phase separation is small in magnitude, disrupting the α-helices decreases phase separation, unlike the α-helices in Whi3 that drive clustering but discourage phase separation (21). The dynamic and transient nature of the helical regions implied by their partial population might facilitate versatile interactions with both helical structures and disordered regions.…”

“…Additionally, the domains studied so far have been essentially disordered without significant population of partial secondary structure and hence do not represent the entire range of transient structures formed by intrinsically disordered domains that phase separate. Yet, previous studies have established the pivotal role of transient α-helical secondary structure in mediating protein self-interaction and phase separation in the case of the human RNA-binding protein TDP-43 (19,20) or where dispersed phase oligomerization of helices opposes phase separation in the case of the glutamine-rich RNAbinding proteins from the multinucleate fungus Ashbya gossypii (21). Hence, it is important both to identify secondary structure in phase separating proteins and evaluate secondary structure contribution to phase separation.…”

Asymmetric oligomerization state and sequence patterning can tune multiphase condensate miscibility

“…Oligomerization can reduce the entropic cost of LLPS by increasing the effective chain length of proteins, thereby functioning as an entropic knob. Alternatively, monomers can also be potentially sequestered away into stable oligomers, which could hinder LLPS 23 . Oligomerization has also been harnessed to allow the formation of de novo condensates [24][25][26][27] .…”