Charge-driven condensation of RNA and proteins suggests broad role of phase separation in cytoplasmic environments

Phase separation processes are increasingly being recognized as important organizing mechanisms of biological macromolecules in cellular environments. Well-established drivers of phase separation are multi-valency and intrinsic disorder. Here, we show that globular macromolecules may condense simply based on electrostatic complementarity. More specifically, phase separation of mixtures between RNA and positively charged proteins is described from a combination of multiscale computer simulations with microscopy and spectroscopy experiments. Phase diagrams were mapped out as a function of molecular concentrations in experiment and as a function of molecular size and temperature via simulations. The resulting condensates were found to retain at least some degree of internal dynamics varying as a function of the molecular composition. The results suggest a more general principle for phase separation that is based primarily on electrostatic complementarity without invoking polymer properties as in most previous studies. Simulation results furthermore suggest that such phase separation may occur widely in heterogenous cellular environment between nucleic acid and protein components.


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• You should state whether an appropriate sample size was computed when the study was being designed • You should state the statistical method of sample size computation and any required assumptions • If no explicit power analysis was used, you should describe how you decided what sample (replicate) size (number) to use Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission:

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Most data in Fig. 4 were measured once, although each measurement observed dozens or hundreds of condensates. Measurements in Fig. 5 were repeated 3 times, as shown in Supplementary Fisg. 26 and 27. Most data in Fig. 6 were measured once but one concentration was measured twice, once exhibiting phase separation and once not exhibiting phase separation. This indicates the reproducibility of creating the concentrations at the boundary between the phases. Data in Fig. 7 were measured once, except for one set of concentrations, indicated by two points, which shows the typical reproducibility.
Individual simulations were not repeated but a wide range of concentrations and conditions were considered, and simulations were extended well beyond typical times to achieve convergence. Moreover, highly consistent results were obtained for similar conditions indicating reproducibility of the results reported here (see for example Figure 2).
Simulation results reported based on particle averages (such as diffusion estimates) benefit from ensemble averages over tens to hundreds of equivalent particles.
The statistical analyses described above do not apply to most of the data in this submission for the reasons described above.
For reported results where statistical analyses are appropriate (such as particleaveraged diffusion estimates from simulation) standard errors of the mean are reported. P-values were not calculated because reported values inside and outside the condensates were grossly different compared to the estimated uncertainties. 3 • Indicate how samples were allocated into experimental groups (in the case of clinical studies, please specify allocation to treatment method); if randomization was used, please also state if restricted randomization was applied • Indicate if masking was used during group allocation, data collection and/or data analysis Please outline where this information can be found within the submission (e.g., sections or figure legends), or explain why this information doesn't apply to your submission: Additional data files ("source data") • We encourage you to upload relevant additional data files, such as numerical data that are represented as a graph in a figure, or as a summary table • Where provided, these should be in the most useful format, and they can be uploaded as "Source data" files linked to a main figure or table • Include model definition files including the full list of parameters used • Include code used for data analysis (e.g., R, MatLab) • Avoid stating that data files are "available upon request" Please indicate the figures or tables for which source data files have been provided: This information does not apply to our submission due to the reasons indicated above.
Quantitatively accurate results are reported in the text. Figures show qualitatively meaningful results and high-resolution image data is provided as supplementary data.