Divalent cations can control a switch-like behavior in heterotypic and homotypic RNA coacervates

Abstract

Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model arginine-rich peptide-RNA system, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a generic mechanism for modulating the biochemical environment of RNA coacervates in a cellular context. Phase separation aids in regulating essential functions of cells and organisms. Within the cellular context, condensation-driven phase separation of biomolecules selectively compartmentalizes essential proteins, nucleic acids, and biochemical processes 1 . Liquid-liquid phase transitions can drive dynamic intracellular compartmentalization to form membrane-less organelles (MLOs) such as the nucleolus and stress granules 2-5 . Unlike their membrane-bound counterparts such as the nucleus, the unique physicochemical features of MLOs can enable liquidlike behavior such as facile formation, fusion, and dissolution 2 . These dynamic qualities provide a means for cells to sense and respond rapidly to changing environments, such as the cytoplasm during stress 2 . In this context, stimulus-dependent liquid-liquid phase separation (LLPS) of biopolymers has recently emerged with numerous implications in biology 6,7 .One of the driving forces of MLO formation is the weak multivalent interactions among proteins and nucleic acids 8 . Quantitative understanding of the underlying interaction network and elucidation of molecular driving forces that alter them are therefore key topics of research. The interchain associations of proteins and nucleic acids are largely determined by their primary sequences 8 . Intrinsically-disordered proteins (IDPs) with low-complexity repetitive sequences have been identified as drivers of MLO biogenesis, with charged sequences being one of the most common motifs found in intracellular MLOs 9 . Phase separation of charged low-complexity motifs are largely driven by electrostatic interactions 8,10,11 . This phenomenon, commonly known as complex coacervation, can be recapitulated in an in vitro model consisting of an arginine-rich peptide and RNA mixtures 12,13 . Our recent work demonstrated that a peptide-RNA mixture can display reentrant phase behavior, where droplets can form and dissolve due to monotonic variation of ionic peptide-RNA ratios alone 12 . This type of behavior can be modeled simply using early...

Zinc and Copper Ions Differentially Regulate Prion-Like Phase Separation Dynamics of Pan-Virus Nucleocapsid Biomolecular Condensates

“…Tight control of divalent ion concentrations and homeostasis is most critical to cellular health and longevity [ 127 ]. Rapidly changing cellular conditions, including stress, cause ion fluxes, coinciding with formation and dissolution of MLOs and peptide-RNA condensates [ 128 ]. Whereas divalent ions, such as Zn 2+ , positively influence phase separation [ 12 ], others (e.g., Mg 2+ , Ca 2+ ) negatively influence PrLD–RNA interactions and reduce LLPS, as does the metal chelator EDTA [ 128 ].…”

Neuronal ribonucleoprotein granules: Dynamic sensors of localized signals

“…How would such a maturation process be spatially regulated? Based on in vitro and in silico work, one could speculate that spatial gradients of neuronal RNP granule maturation may reflect gradients of molecules regulating phase behavior, such as posttranslational modifiers, divalent cations or ATP . As described further in the next section, local and specific disassembly or remodeling of RNP granules in response to environmental signals (eg, synaptic activity or guidance molecules) may also lead to local changes in soluble protein/RNA concentrations, and RNP granule composition.…”

“…Although physiological evidence is still lacking, PTM‐independent switch mechanisms are likely to also be at play in neuronal cells. For example, changes in cation concentration or pH have been reported in distinct neuronal compartments in response to local stimuli, and were shown to dramatically modulate phase behavior in vitro . Variations in Ca 2+ concentration, in particular, induce a switch‐like behavior between heterotypic and homotypic RNA droplets in vitro, thus dictating component partitioning and droplet composition …”

Dynamic Synthetic Cells Based on Liquid–Liquid Phase Separation

“…The addition of inorganic salts has commonlyb een used to trigger disassembly of droplets formed by complex coacervation between oppositely charged polyelectrolytes, due to the decreased entropyg ain associated with release of counter-ions above ac riticals alt concentration. [103] However, the addition of salts is hard to reverse. [103] However, the addition of salts is hard to reverse.…”

“…[26,102] Interestingly,arecents tudy reported switch-like behaviour in RNA-based droplets in response to divalent cations, with heterotypico rh omotypic droplets being formed depending on the concentrationo fm agnesium ions. [103] However, the addition of salts is hard to reverse. The use of stimulus-responsive species capable of phase separation throughc hanges in the solution parameters,s uch as the pH or temperature, offers ac leara dvantagef or achieving reversible control over phase separation.…”