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Phase Transition of RNA−Protein Complexes Into Ordered Hollow Condensates

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Phase Transition of RNA−Protein Complexes Into Ordered Hollow Condensates Phase transition of RNA−protein complexes into ordered hollow condensates Ibraheem Alshareedaha,1, Mahdi Muhammad Moosaa,1, Muralikrishna Rajub, Davit A. Potoyanb,2, and Priya R. Banerjeea,2 aDepartment of Physics, University at Buffalo, The State University of New York, Buffalo, NY 14260; and bDepartment of Chemistry, Iowa State University, Ames, IA 50011 Edited by David A. Weitz, Harvard University, Cambridge, MA, and approved May 27, 2020 (received for review December 20, 2019) Liquid−liquid phase separation of multivalent intrinsically disor- morphologies (14–16).Inanothersystem,itwasobservedthat dered protein−RNA complexes is ubiquitous in both natural and simple overexpression of TDP-43, a stress granule protein, biomimetic systems. So far, isotropic liquid droplets are the most can give rise to multilayered compartments with vacuolated commonly observed topology of RNA−protein condensates in ex- nucleoplasm-filled internal space (17). However, the physical periments and simulations. Here, by systematically studying the driving forces behind these hollow morphologies remain phase behavior of RNA−protein complexes across varied mixture poorly understood. compositions, we report a hollow vesicle-like condensate phase of Recently, we demonstrated that RNA can mediate a reentrant nucleoprotein assemblies that is distinct from RNA−protein drop- phase transition of ribonucleoproteins (RNPs) containing arginine- lets. We show that these vesicular condensates are stable at specific rich low-complexity domains (LCDs) through multivalent heterotypic mixture compositions and concentration regimes within the phase interactions (18, 19). At the substoichiometric regime, RNA triggers diagram and are formed through the phase separation of anisotropic RNP phase separation, whereas, at superstoichiometric ratios, excess protein−RNA complexes. Similar to membranes composed of am- RNA leads to droplet dissolution due to charge inversion on the phiphilic lipids, these nucleoprotein−RNA vesicular membranes ex- surface of RNP−RNA complexes (18). This inversion of charge hibit local ordering, size-dependent permeability, and selective suggests that the stoichiometry of these fuzzy protein−RNA as- encapsulation capacity without sacrificing their dynamic formation semblies is not fixed but varies with the mixture composition. The and dissolution in response to physicochemical stimuli. Our findings condensate dissolution at superstoichiometric ratios is expected to suggest that protein−RNA complexes can robustly create lipid-free be generic for systems with obligate heterotypic interactions (20). BIOPHYSICS AND COMPUTATIONAL BIOLOGY vesicle-like enclosures by phase separation. Interestingly, we also observed that sudden jumps in RNA-to-LCD stoichiometry can lead to the formation of transient hollow droplet RNA vesicles | biomolecular condensates | MD simulation | topologies under nonequilibrium conditions (18). Although these optical tweezer | nucleoprotein assembly vacuolated structures were observed only as fleeting intermediates (mean lifetime < 300 s) during the reentrant dissolution of protein−RNA recise spatiotemporal control of biochemical processes is condensates (18), their occurrence nevertheless suggested that Pindispensable to life. Classically, it was assumed that am- associative RNA−protein systems may have access to complex phiphilic lipids provide living systems the capacity to segregate morphologies distinct from isotropic liquid droplets. This leads to different biological processes into distinct membrane-bound compartments and therefore afford spatial and temporal sepa- Significance ration of (sub)cellular biochemistry (1). These membrane-bound compartments provide an independent internal environment Vesicular assemblies play crucial roles in the subcellular orga- that can be tuned as per cellular needs. Recent advances nization as well as in biotechnological applications. Classically, indicate that, in addition to membrane-bound organelles, the ability to form such assemblies was primarily assigned to cells also utilize membrane-free protein/RNA-rich conden- lipids and lipid-like amphiphilic molecules. Here, we show that sates as compartments for organizing the intracellular space disordered RNA−protein complexes can form vesicle-like ordered (2–5). These fluid compartments, often referred to as mem- assemblies at disproportionate mixture compositions. We also braneless organelles (MLOs), are formed by liquid−liquid phase show that the ability to form vesicular assemblies is generic to separation (LLPS) and can partition a diverse set of biomolecules multicomponent systems where phase separation is driven by selectively (6). One key advantage of these MLOs is their dynamic heterotypic interactions. We speculate that such vesicular as- nature wherein they can rapidly form and dissolve in a stimuli- semblies play crucial roles in the formation of dynamic multilay- responsive fashion. ered subcellular membraneless organelles and can be utilized to Since many MLOs are thought to form by LLPS, their internal fabricate novel stimuli-responsive microscale systems. microenvironment is significantly higher in density as compared to the surrounding cytoplasm/nucleoplasm and exhibits complex Author contributions: M.M.M. and P.R.B. designed research; M.R. and D.A.P. designed the fluidlike properties (7). To spatially segregate biochemical pro- molecular dynamics simulation; I.A., M.M.M., M.R., D.A.P., and P.R.B. performed research; I.A., M.R., D.A.P., and P.R.B. analyzed data; and I.A., M.M.M., and P.R.B. wrote the paper. cesses within this condensed phase, many MLOs [e.g., nuclei (8), Competing interest statement: P.R.B, M.M.M., and I.A. have a pending patent application nuclear speckles (9), paraspeckles (10), stress granules (11, 12), related to the present study: “Lipid-free polyionic vesicles and methods of making and and P granules (13)] utilize distinct subcompartments within using same,” provisional application US 62/958,039 filed by University at Buffalo, The themselves. Such multilayered structures are best described State University of New York, January 2020. by a coexisting multiphasic condensate model where two or This article is a PNAS Direct Submission. more distinct types of condensates are formed by LLPS of Published under the PNAS license. individual components in a multicomponent mixture (5). 1I.A. and M.M.M. contributed equally to this work. However, a different class of multilayered MLOs has also 2To whom correspondence may be addressed. Email: [email protected] or prbanerj@ been reported, where the layered topology is manifested due buffalo.edu. to the presence of a hollow internal space. For example, This article contains supporting information online at https://www.pnas.org/lookup/suppl/ in vivo experiments have demonstrated that both nuclear and doi:10.1073/pnas.1922365117/-/DCSupplemental. cytoplasmic germ granules of Drosophila can exhibit hollow www.pnas.org/cgi/doi/10.1073/pnas.1922365117 PNAS Latest Articles | 1of9 Downloaded by guest on September 29, 2021 the following question: Are there specific regions within the phase Results and Discussion diagram of RNA−protein mixtures where such multilayered Nucleoprotein−RNA Complexes Form Stable Hollow Condensates structures form spontaneously? with Vesicle-Like Properties. Our previous observations suggested Here, to ascertain this possibility, we explore the liquid−liquid that hollow condensate topology may exist when RNA concen- phase separation regime within protein−RNA reentrant phase tration is substantially higher than RNP concentration (18). To space that spans three orders of magnitude in concentrations. investigate such complex morphologies, we first probed for the Using a combination of biophysical and computational tools (e.g., mesoscale structure of condensates formed by a model arginine- SI Appendix confocal microscopy, ensemble spectroscopy, optical tweezers, rich disordered nucleoprotein, protamine (PRM) ( , Fig. S1A and ref. 21), with a single-stranded RNA [poly(U)] at microfluidic manipulation, and molecular dynamics [MD] simu- C = × C lation), we report a hitherto unknown structural transition from excess RNA conditions ( RNA 5 PRM). Remarkably, we observed that micrometer-sized hollow condensates are readily isotropic liquid droplets to a vesicle-like phase in nucleoprotein−RNA formed by mixing 4.4 mg/mL PRM with 22 mg/mL RNA. These condensates. These vesicle-like condensates are hollow struc- hollow condensates closely resemble lipid vesicles in their ap- − tures enclosed by an ordered nucleoprotein RNA membrane, pearance (Fig. 1A). Furthermore, we observed that these hollow − and are formed through liquid liquid phase separation of condensates are relatively stable across a wide range of tem- nucleoprotein−RNA complexes at distinct mixture compositions perature and salt concentrations (SI Appendix, Fig. S2). Below ([nucleotide]:[Arg] is approximately either >1.87 or <0.075) and 11 mg/mL RNA, PRM−RNA mixtures formed spherical liquid concentration regimes. droplets that are uniform in density (Fig. 1 A and B and SI Fig. 1. PRM−RNA mixtures can form vesicular assemblies. (A) Fluorescence and differential interference contrast (DIC) micrographs of PRM−RNA vesicles formed at 4.4 mg/mL PRM and 22 mg/mL poly(U) RNA (i.e., [PRM] = 5 × [RNA]; Left). Contrastingly, at a lower PRM-to-RNA ratio, PRM−RNA mixtures form isotropic liquid droplets. Shown in Right are micrographs of samples prepared upon mixing 4.4 mg/mL PRM and 2.2 mg/mL poly(U) RNA
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