Phase separation of multivalent protein and RNA molecules underlies the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that typically organize into multilayered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we deploy systematic experiments and simulations based on coarse-grained models to show that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multilayered condensates with distinct material properties. These studies yield a set of rules regarding homotypic and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

Collective phase transitions, including phase separation and gelation of multivalent protein and RNA molecules appears to underlie the biogenesis of biomolecular condensates such as membraneless organelles. In vivo, these condensates encompass hundreds of distinct types of molecules that are often organized into multi-layered structures supporting the differential partitioning of molecules into distinct regions with distinct material properties. The interplay between driven (active) versus spontaneous (passive) processes that are required for enabling the formation of condensates with coexisting layers of distinct material properties remains unclear. Here, we investigate the role of spontaneous driving forces as determinants of protein-RNA condensates with complex morphologies and distinct material properties. Through the use of systematic in vitro experiments and simulations based on coarse-grained models we find that that the collective interactions among the simplest, biologically relevant proteins and archetypal RNA molecules are sufficient for driving the spontaneous emergence of multi-layered condensates with distinct material properties. Our results demonstrate that key properties of protein-RNA condensates such as their overall morphologies, internal dynamics, and the selective partitioning of substrates are governed specific amino acid chemistries as well as RNA sequence and secondary structure. Our findings yield a clear set of heuristics regarding homo- and heterotypic interactions that are likely to be relevant for understanding the interplay between active and passive processes that control the formation of functional biomolecular condensates.

Understanding the kinetics, thermodynamics, and molecular mechanisms of liquid–liquid phase separation (LLPS) is of paramount importance in cell biology, requiring reproducible methods for studying often severely aggregation-prone proteins. Frequently applied approaches for inducing LLPS, such as dilution of the protein from an urea-containing solution or cleavage of its fused solubility tag, often lead to very different kinetic behaviors. Here we demonstrate that at carefully selected pH values proteins such as the low-complexity domain of hnRNPA2, TDP-43, and NUP98, or the stress protein ERD14, can be kept in solution and their LLPS can then be induced by a jump to native pH. This approach represents a generic method for studying the full kinetic trajectory of LLPS under near native conditions that can be easily controlled, providing a platform for the characterization of physiologically relevant phase-separation behavior of diverse proteins.