![]() In this context, the development of generic methods, integrating the knowledge accumulated from phase separation in vitro studies, would be particularly acute to elucidate the general principles of the structuring role of RNA within a condensed phase in the cellular environment. Moreover, RNA appears to determine the specificity of the molecular composition of the granules as shown for polyQ-dependent RNA–protein assemblies 24. ![]() More recently, it has been proposed that defects in nuclear RNA levels lead to excessive phase separation of IDR-containing RNA-binding proteins (RBPs) such as FUS and TDP-43 23. For instance, the polar positioning of germ granules found in Caenorhabditis elegans reflects an RNA competition mechanism that regulates local phase separation 19, 20, while rRNA transcription allows the cells to overcome the inherent stochastic nature of phase separation by timely seeding of nucleolus assembly in Drosophila melanogaster embryos 21, 22. By acting as a molecular seed, RNA contributes to the spatiotemporal regulation of phase-separated granules 17, 18. In vitro studies identified that RNA modulates the biophysical properties of liquid droplets, by tuning their viscosity and their dynamics 15, 16. RNAs can promote phase separation synergistically with protein–protein interactions but also independently 13, 14. However, new roles of RNA in the regulation of granule formation, which has been long assigned to protein components, are being uncovered 12. A general model has emerged where RNP granules generate from liquid–liquid phase separation, driven by low-affinity interactions of multivalent proteins and/or proteins containing intrinsically disordered regions (IDRs) 7, 8, 9, 10, 11. RNP granules behave as highly concentrated liquid-like condensates that rapidly exchange components with the surrounding medium 3, 4 and their formation relies on a self-assembly process 5, 6. Although RNP granules exhibit different compositions and functions depending on the cellular context, they have strikingly common features concerning their biophysical behavior and assembly process. They regulate RNA processing and thereby play a pivotal role in overall gene expression output, whereas their dysfunction is linked to viral infection, cancer, and neurodegenerative diseases 1, 2. Among them, ribonucleoprotein (RNP) granules, which include processing bodies (P-bodies), stress granules (SGs), germ granules, nucleoli, Cajal bodies, etc., are supramolecular assemblies of RNA molecules and proteins found in eukaryotic cells 1. Membrane-less organelles, by localizing and regulating complex biochemical reactions, are ubiquitous functional subunits of intracellular organization. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. ![]() We demonstrate that intracellular RNA seeds the nucleation of the condensates, as it provides molecular cues to locally coordinate the formation of endogenous high-order RNP assemblies. Here we report the versatile scaffold ArtiG to generate concentration-dependent RNA–protein condensates within living cells, as a bottom-up approach to study the impact of co-segregated endogenous components on phase separation. Numerous in vitro approaches have validated this model, yet a missing aspect is to take into consideration the complex molecular mixture and promiscuous interactions found in vivo. Liquid–liquid phase separation is thought to be a key organizing principle in eukaryotic cells to generate highly concentrated dynamic assemblies, such as the RNP granules.
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