Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (Rmotifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

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. Here 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 RNA concentration initially leads to droplet assembly via 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.

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.

Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context.

Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model arginine-rich peptide-RNA system, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a generic mechanism for modulating the biochemical environment of RNA coacervates in a cellular context. Phase separation aids in regulating essential functions of cells and organisms. Within the cellular context, condensation-driven phase separation of biomolecules selectively compartmentalizes essential proteins, nucleic acids, and biochemical processes 1 . Liquid-liquid phase transitions can drive dynamic intracellular compartmentalization to form membrane-less organelles (MLOs) such as the nucleolus and stress granules 2-5 . Unlike their membrane-bound counterparts such as the nucleus, the unique physicochemical features of MLOs can enable liquidlike behavior such as facile formation, fusion, and dissolution 2 . These dynamic qualities provide a means for cells to sense and respond rapidly to changing environments, such as the cytoplasm during stress 2 . In this context, stimulus-dependent liquid-liquid phase separation (LLPS) of biopolymers has recently emerged with numerous implications in biology 6,7 .One of the driving forces of MLO formation is the weak multivalent interactions among proteins and nucleic acids 8 . Quantitative understanding of the underlying interaction network and elucidation of molecular driving forces that alter them are therefore key topics of research. The interchain associations of proteins and nucleic acids are largely determined by their primary sequences 8 . Intrinsically-disordered proteins (IDPs) with low-complexity repetitive sequences have been identified as drivers of MLO biogenesis, with charged sequences being one of the most common motifs found in intracellular MLOs 9 . Phase separation of charged low-complexity motifs are largely driven by electrostatic interactions 8,10,11 . This phenomenon, commonly known as complex coacervation, can be recapitulated in an in vitro model consisting of an arginine-rich peptide and RNA mixtures 12,13 . Our recent work demonstrated that a peptide-RNA mixture can display reentrant phase behavior, where droplets can form and dissolve due to monotonic variation of ionic peptide-RNA ratios alone 12 . This type of behavior can be modeled simply using early...