Interplay between Short-Range Attraction and Long-Range Repulsion Controls Reentrant Liquid Condensation of Ribonucleoprotein–RNA Complexes

Abstract

In eukaryotic cells, ribonucleoproteins (RNPs) form mesoscale condensates by liquid–liquid phase separation that play essential roles in subcellular dynamic compartmentalization. The formation and dissolution of many RNP condensates are finely dependent on the RNA-to-RNP ratio, giving rise to a windowlike phase separation behavior. This is commonly referred to as reentrant liquid condensation (RLC). Here, using ribonucleoprotein-inspired polypeptides with low-complexity RNA-binding sequences as well as an archetypal disordered RNP, fused in sarcoma, as model systems, we investigate the molecular driving forces underlying this nonmonotonous phase transition. We show that an interplay between short-range cation−π attractions and long-range electrostatic forces governs the heterotypic RLC behavior of RNP–RNA complexes. Short-range attractions, which can be encoded by both polypeptide chain primary sequence and nucleic acid base sequence, control the two-phase coexistence regime, regulate material properties of polypeptide–RNA condensates, and oppose condensate reentrant dissolution. In the presence of excess RNA, a competition between short-range attraction and long-range electrostatic repulsion drives the formation of a colloidlike cluster phase. With increasing short-range attraction, the fluid dynamics of the cluster phase is arrested, leading to the formation of a colloidal gel. Our results reveal that phase behavior, supramolecular organization, and material states of RNP–RNA assemblies are controlled by a dynamic interplay between molecular interactions at different length scales.

Prebiotically-relevant low polyion multivalency can improve functionality of membraneless compartments

“…We attribute the surprisingly high salt stability of (Arg)10/nucleotide coacervates to cation-pi binding, which is known to be strong for Arg residues and adenosine nucleobases. 25,26,28 Early Earth conditions are thought to have encompassed a wide range of salt concentrations, from ponds to ocean water. 29,30 Our results indicate that even at just n=10, certain oligopeptidebased coacervates persist even above 1 M ionic strength, supporting the relevance of coacervatebased prebiotic compartments to diverse prebiotic scenarios extending beyond freshwater to brackish waters, oceans or submarine hydrothermal vent systems alike.…”

Sequence-encoded and Composition-dependent Protein-RNA Interactions Control Multiphasic Condensate Morphologies

“…1d). Such phase behavior is usually referred to as reentrant phase transition 28,47,49 . Within the reentrant phase separation window ( Fig.…”

“…To experimentally test this idea, we designed an all K-variant of RLD (KLD: [KGKGG]5, Table S2) that selectively weakens the PLD-RLD interactions while preserving the ability to phase separate with RNA. The R-to-K substitution is designed based on prior studies showing that (i) lysine residues have a much lesser potency (as compared to arginine residues) to interact with tyrosine and other -rich amino acids/nucleobases 21,28,29,46,57,58 , and (ii) KLDs can phase separate with RNA via ionic interactions since lysine is expected to carry a similar charge to that of arginine 28 . Indeed, our state diagram analysis indicates that KLD has no impact on PLD phase separation, even at KLD concentrations that are 20 times more than the PLD concentration (Fig 5a).…”

“…Analysis of sequence features of eukaryotic BMC proteins revealed that intrinsically disordered low complexity domains (LCDs) are common drivers and/or regulators of RNP phase separation with and without RNAs [19][20][21][22][23] . The LCD sequence composition and patterning provide programmable modules for dynamic multivalent protein-protein and protein-RNA interactions [24][25][26][27][28][29] . In multi-component mixtures, these dynamic inter-chain interactions are ubiquitous and can either cooperate or compete within a dense network of LC proteins and RNAs 30,31 .…”

“…These R-rich repeat polymers can invade SGs and impair their fluid dynamics by aberrantly interacting with SG components, including PLDs and RNAs 29,[42][43][44] . Within the PLD-RLD-RNA system, three interactions have the capacity to drive independent phase separation processes: PLD-PLD interactions can drive homotypic PLD phase separation 23,45 , PLD-RLD interactions can drive the co-phase separation of PLD-RLD into heterotypic condensates 46 , and RLD-RNA interactions can drive the formation of RLD-RNA condensates 28,47,48 . Therefore, the RLDs within the PLD-RLD-RNA system represent promiscuous modules that can interact with both PLDs and RNAs 42,43,46 .…”