A crucial feature of differentiated cells is the rapid activation of enhancer-driven transcriptional programs in response to signals. The potential contributions of physicochemical properties of
Multivalent protein-protein and protein-RNA interactions are the drivers of biological phase separation. Biomolecular condensates typically contain a dense network of multiple proteins and RNAs, and their competing molecular interactions play key roles in regulating the condensate composition and structure. Employing a ternary system comprising of a prion-like polypeptide (PLP), arginine-rich polypeptide (RRP), and RNA, we show that competition between the PLP and RNA for a single shared partner, the RRP, leads to RNA-induced demixing of PLP-RRP condensates into stable coexisting phases—homotypic PLP condensates and heterotypic RRP-RNA condensates. The morphology of these biphasic condensates (non-engulfing/ partial engulfing/ complete engulfing) is determined by the RNA-to-RRP stoichiometry and the hierarchy of intermolecular interactions, providing a glimpse of the broad range of multiphasic patterns that are accessible to these condensates. Our findings provide a minimal set of physical rules that govern the composition and spatial organization of multicomponent and multiphasic biomolecular condensates.
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.
Liquid-liquid phase separation of multivalent proteins and RNAs drives the formation of biomolecular condensates that facilitate membrane-free compartmentalization of subcellular processes. With recent advances, it is becoming increasingly clear that biomolecular condensates are network fluids with time-dependent material properties. Here, employing microrheology with optical tweezers, we reveal molecular determinants that govern the viscoelastic behavior of condensates formed by multivalent Arg/Gly-rich sticker-spacer polypeptides and RNA. These condensates behave as Maxwell fluids with an elastically-dominant rheological response at shorter timescales and a liquid-like behavior at longer timescales. The viscous and elastic regimes of these condensates can be tuned by the polypeptide and RNA sequences as well as their mixture compositions. Our results establish a quantitative link between the sequence- and structure-encoded biomolecular interactions at the microscopic scale and the rheological properties of the resulting condensates at the mesoscale, enabling a route to systematically probe and rationally engineer biomolecular condensates with programmable mechanics.
Liquid−liquid phase separation of multivalent intrinsically disordered protein−RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA−protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA−protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA−protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein−RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein−RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein−RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.
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