Phase transition of RNA-protein complexes into ordered hollow condensates

Abstract

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.

Keywords: MD simulation; RNA vesicles; biomolecular condensates; nucleoprotein assembly; optical tweezer.

Fig. 1.

PRM−RNA mixtures can form vesicular assemblies. (A) Fluorescence and differential interference contrast (DIC) micrographs of PRM−RNA vesicles formed at 4.4 mg/mL PRM and 22 mg/mL poly(U) RNA (i.e., [PRM] = 5 × [RNA]; Left). Contrastingly, at a lower PRM-to-RNA ratio, PRM−RNA mixtures form isotropic liquid droplets. Shown in Right are micrographs of samples prepared upon mixing 4.4 mg/mL PRM and 2.2 mg/mL poly(U) RNA (i.e., [PRM] = 0.5 × [RNA]). (B) Fluorescence micrographs showing that mixture composition governs PRM−RNA droplets and vesicles formation. PRM concentration was fixed at 4.4 mg/mL. (C) A 3D reconstruction of a PRM−RNA vesicle. (D) RNA (probed using SYTO13) and PRM (probed using Alexa594-labeled PRM) localize within the vesicle rim, while the lumen has a hollow appearance. The fluorescence intensity of the lumen is similar to the external dilute phase. (E) FCS autocorrelation curves for freely diffusing TMR-labeled Dextran-4.4k (molecular weight 4.4 kDa) in three regions: inside the lumen, within the rim, and outside the hollow condensate. This experiment was done by focusing the confocal volume inside a hollow condensate, outside and on the rim (SI Appendix, Fig. S6 and SI Materials and Methods). These autocorrelation curves suggest macromolecular diffusion is significantly slowed down (autocorrelation decays at ∼100 ms) within the rim as compared to the lumen and the external dilute phase (autocorrelation decays at ∼100 µs). (F) FRAP images and the corresponding intensity time trace for PRM-A594 showing nearly complete fluorescence recovery of the hollow condensate rim. The yellow circle indicates the bleaching region (Movie S1). (G) Optical tweezer-controlled fusion of two PRM−RNA hollow condensates (Top) and a PRM−RNA droplet with a hollow condensate (Bottom) (see also corresponding Movies S2 and S3, respectively). Experiments were performed in 25 mM Tris⋅HCl buffer (pH 7.5). Fluorescent probe concentrations were ≤1% of the unlabeled protein and RNA. Fluorescence microscopy was performed with Alexa594-labeled PRM unless otherwise noted. (Scale bars, 10 µm.)

Fig. 2.

Vesicle-like polypeptide−RNA condensate is a thermodynamically stable phase. (A) Thermodynamic state diagram of PRM−poly(U) mixture shows three distinct phases: a homogeneous state (filled gray circles), PRM-poly(U) isotropic liquid droplets (filled blue circles), and PRM-poly(U) vesicles (open blue circles). The dashed line represents the boundary between homogeneous and phase-separated regimes. The background colored shade represents the estimated concentration of electric charge (calculated from mixture composition). Hollow condensates were present at two narrow regimes within this state diagram, as indicated by dotted lines. The dashed and dotted lines are drawn as guides to the eye. Condensate imaging was done using PRM-A594. (B) Electrophoretic mobility measured by dynamic light scattering of PRM−poly(U) condensates as a function of poly(U)-to-PRM ratio. These data clearly show charge inversion: Condensates have a net positive charge at low RNA-to-PRM ratio and a net negative charge at high RNA-to-PRM ratio. PRM concentration was 1.1 mg/mL for this experiment. (C) Fluorescence micrographs of hollow condensates at RNA excess (upper edge of the LLPS region in A) and PRM excess (lower edge of the LLPS region in A) conditions. The excess PRM sample was prepared at 8.8 mg/mL PRM and 0.44 mg/mL poly(U). The excess RNA sample was made at 4.4 mg/mL PRM and 22 mg/mL poly(U). (D) A scheme showing droplet-to-vesicle and vesicle-to-droplet transformations upon RNA influx and removal, respectively (Middle). Differential interference contrast (DIC) images of a PRM−RNA droplet [0.88 mg/mL PRM, 0.44 mg/mL poly(U)] transitioning to a vesicle upon RNA influx (Top; also see SI Appendix, Fig. S11 for experimental details; Movie S4). DIC images of a PRM−RNA vesicle transitioning to a homogeneous droplet as a result of RNA removal (RNase-A treatment; Bottom; see also Movie S5). (E and F) Fluorescence micrographs (Top) and corresponding intensity profiles (Bottom) of (E) hollow condensates formed by PRM-K (4.4 mg/mL) and poly(U) RNA (22 mg/mL), and (F) FUS^RGG3 (4.0 mg/mL) and poly(U) RNA (20 mg/mL). (Scale bars, 10 µm.)

Fig. 3.

Intracomplex disproportionation drives the formation of vesicular assemblies. (A) A scheme for the formation of RNA−protein hollow condensates. Left shows representative experimental observations (fluorescence micrographs). The proposed mechanism is shown in Middle and Right. At low concentrations, nucleoprotein−RNA mixtures form tadpole-like complexes (Top). Increasing the total concentration leads to the formation of small spherical micellar assemblies (Middle). At a relatively high total concentration, nucleoprotein−RNA vesicular structures are formed (Bottom). (Scale bars, 20 µm.) (B) Schematic representation of the protein and RNA chains and the interaction potential employed in our MD simulations. (C) Equilibrium MD configurations showing tadpole, micelle, and vesicle formed under charge disproportionate conditions, C_RNA = 5 × C_PRM. Also see SI Appendix, Fig. S13. (D) Density profiles of PRM and RNA chains for the vesicle obtained from the MD trajectory. See SI Appendix, Fig. S5 for experimental data. (E) Diffusion trajectories of a tagged particle (red sphere) located within the lumen, within the rim, and outside of the vesicle (SI Appendix, Fig. S14).

Fig. 4.

Hollow condensates are widely observed in various biological and synthetic ternary systems. Fluorescence and differential interference contrast (DIC) micrographs of hollow condensates formed by (A) PRM (4.4 mg/mL) and polyP (22.0 mg/mL), (B) PRM (17.6 mg/mL) and poly (E) (18 mg/mL), (C) PRM (8.8 mg/mL) and PAA (22 mg/mL), (D) PAH (40 mg/mL) and poly(U) (4.0 mg/mL), (E) PAH (70 mg/mL) and polyP (4 mg/mL), and (F) [RGRGG]₅ (0.024 mg/mL) and cellular RNA (8.9 mg/mL). See also SI Appendix, Fig. S17. (Scale bars, 10 µm.)

Fig. 5.

PRM−RNA hollow condensate rims exhibit classical lipid-bound membrane-like properties. (A) Optical images of PRM−RNA hollow condensates with cross-polarizing light show birefringence, indicating molecular ordering in the vesicle rims. Corresponding fluorescence micrographs are also shown. (B) Fluorescence images and corresponding intensity profiles of TMR-labeled dextran probes of different molecular weights reveal size-dependent partitioning in PRM−RNA hollow condensates. The low-molecular-weight dextran, Dex-4.4k, partitions to the rim, whereas the high-molecular-weight dextran, Dex-155k (molecular weight 155 kDa), remains excluded from the rim. See also SI Appendix, Fig. S20. (C) A bar plot of size-dependent partitioning data of dextrans into the rim and lumen of PRM−RNA hollow condensates. (D) Partitioning of nucleic acids and proteins into PRM−RNA vesicles. The intensity profiles are measured along a horizontal line passing through the center of individual hollow condensates. (E) Inclusion coefficients (defined as the ratio of mean intensity in the lumen to mean intensity in the external dilute phase) are shown. The statistics were estimated using a minimum of 50 different hollow condensates per sample. Individual points are shown as gray filled circles. (Scale bars, 10 µm.)