Reentrant Phase Transition Drives Dynamic Substructure Formation in Ribonucleoprotein Droplets

Abstract

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles.

Keywords: electrostatic interactions; intrinsically disordered proteins; membrane-less organelles; phase transitions; vacuolated coacervates.

Figure 1. Reentrant phase transition in the mixtures of synthetic peptides containing R-rich linear motifs and RNA

(a) and (b) Phase boundary curves for RP₃/polyU and SR₈/polyU mixtures, respectively, as determined by the solution turbidity. [peptide] = 200 μM in both cases. (c) Representative confocal fluorescence microscopy images of RP₃/polyU mixtures that correspond to the three distinct regions of the phase diagram. (d) Projection of RP₃/polyU phase diagram on the two-component concentration plane. The dotted line is the locus of the solution turbidity maxima (C₀). Also highlighted are regime-I, II, and III. The color scale represents measured turbidity values at 350 nm. The corresponding turbidity plots are shown in Fig. S1a.

Figure 2. Electrostatic interactions and charge inversion drive the reentrant phase behavior of peptide/RNA systems

(a) Solution turbidity plots as a function of increasing [NaCl] (Also see Fig. S5) showing the effects of charge screening on the phase separation of the mixture. (b) Predicted RP₃/polyU phase diagram using a charge inversion model (see SI Note-1). (c) Experimental detection of charge inversion at C₀. Shown here is an overlay of electrophoretic mobility (red) and solution turbidity as a measure of LLPS (blue) ([RP₃] = 200 μM in 10 mM Tris-HCl, pH 7.9 in all datasets).

Figure 3. Dynamic droplet substructure formation during RNA-mediated mixing phase transition of RNP coacervate droplets

(a)–(c) Confocal fluorescence microscopy images at different time-points of RP₃/polyU droplets (1:0.5 wt/wt; 2 mM RP₃) after injection of additional 0.75 equivalents of polyU (wt/wt) into the bulk phase. Images represent formation, fusion, and expulsion of vacuoles within RP₃/polyU droplets. The corresponding video is shown in Movie-4. (d) Surface rendering of a z-stack confocal fluorescence microscopy image showing an internal vacuole within a RP₃/polyU droplet. White lines represent cross-sections of the image within the vacuole (i), at the edge of the vacuole (ii), and above the vacuole (iii). (e) Fluorescence intensity plots at cross sections i, ii, and iii. The white arrows represent the distance axis (length = 14 μm). The corresponding 2D slices are shown to the right.

Figure 4. Formation and dissolution of RP₃/RNA granules by in vitro transcription

(a) Schematic representation of reentrant phase transition in R-rich linear motif (RLM) systems by RNA synthesis in situ. RNA transcription by T7 RNA polymerase in presence of RLMs leads to the formation of phase-separated RLM/RNA droplets, while further increase in [RNA] by transcription leads to a subsequent mixing phase transition and dissolves the RLM/RNA droplets. Experimental data: (b) Confocal fluorescence and DIC microscopy images at different time-points of RP₃/T7 transcription mixture. [RP₃] = 1 mM. (c) Confocal fluorescence microscopy images of RP₃/polyU granules (1:2 wt/wt) at different time-points after incubation with T7 transcription mixture. [RP₃] = 200 μM. Control measurements in both (a) and (b) at the 60 minute time point of the mixture lacking the polymerase looked very similar to the respective images corresponding to time = 0.

Figure 5. Reentrant phase transition and dynamic droplet substructure formation in the FUS/RNA system

(a) and (b) Phase boundary curves for FUS with polyU and U₄₀ RNA, respectively (determined by measuring the solution turbidity). The critical concentrations (C₁ and C₂) and the boundary conditions (C₀) are shown by vertical dotted lines, while three distinct regimes were labeled in the phase diagram. [FUS] = 10 μM in both cases. (c) DIC and confocal fluorescence microscopy images of FUS/polyU droplets (1:0.03 wt/wt) in regime-II. (d) RNA-mediated vacuole formation within FUS/polyU droplets (final $\frac{[\text{RNA}]}{[\text{Peptide}]}=1:0.05$; wt/wt) using confocal fluorescence and DIC microscopy. Also, see Movie-6.