Proximity editing to identify RNAs in phase-separated RNA binding protein condensates.
Ontology highlight
ABSTRACT:
SUBMITTER: Zhou G
PROVIDER: S-EPMC8382829 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description: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. 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. 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. 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. Interestingly, the co-segregation of intracellular components ultimately impacts the size of the phase-separated condensates. Thus, RNA arises as an architectural element that can influence the composition and the morphological outcome of the condensate phases in an intracellular context.
| S-EPMC6642089 | biostudies-literature
Project description:An emerging mechanism for intracellular organization is liquid-liquid phase separation (LLPS). Found in both the nucleus and the cytoplasm, liquidlike droplets condense to create compartments that are thought to promote and inhibit specific biochemistry. In this work, a multiphase, Cahn-Hilliard diffuse interface model is used to examine RNA-protein interactions driving LLPS. We create a bivalent system that allows for two different species of protein-RNA complexes and model the competition that arises for a shared binding partner, free protein. With this system we demonstrate that the binding and unbinding of distinct RNA-protein complexes leads to diverse spatial pattern formation and dynamics within droplets. Both the initial formation and transient behavior of spatial patterning are subject to the exchange of free proteins between RNA-protein complexes. This study illustrates that spatiotemporal heterogeneity can emerge within phase-separated biological condensates with simple binding reactions and competition. Intradroplet patterning may influence droplet composition and, subsequently, cellular organization on a larger scale.
| S-EPMC6739070 | biostudies-literature
Project description:Stress granules (SGs) are non-membranous organelles facilitating stress responses and linking the pathology of age-related diseases. In a genome-wide imaging-based phenomic screen, we identify Pab1 co-localizing proteins under 2-deoxy-D-glucose (2-DG) induced stress in Saccharomyces cerevisiae. We find that deletion of one of the Pab1 co-localizing proteins, Lsm7, leads to a significant decrease in SG formation. Under 2-DG stress, Lsm7 rapidly forms foci that assist in SG formation. The Lsm7 foci form via liquid-liquid phase separation, and the intrinsically disordered region and the hydrophobic clusters within the Lsm7 sequence are the internal driving forces in promoting Lsm7 phase separation. The dynamic Lsm7 phase-separated condensates appear to work as seeding scaffolds, promoting Pab1 demixing and subsequent SG initiation, seemingly mediated by RNA interactions. The SG initiation mechanism, via Lsm7 phase separation, identified in this work provides valuable clues for understanding the mechanisms underlying SG formation and SG-associated human diseases.
| S-EPMC9240020 | biostudies-literature
Project description:Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.
| S-EPMC6322363 | biostudies-literature
Project description:Cooperation between DNA, RNA and protein regulates gene expression and controls differentiation through interactions that connect regions of nucleic acids and protein domains and through the assembly of biomolecular condensates. Here, we report that endoderm differentiation is regulated by the interaction between the long non-coding RNA (lncRNA) DIGIT and the bromodomain and extraterminal domain protein BRD3. BRD3 forms phase-separated condensates of which the formation is promoted by DIGIT, occupies enhancers of endoderm transcription factors and is required for endoderm differentiation. BRD3 binds to histone H3 acetylated at lysine 18 (H3K18ac) in vitro and co-occupies the genome with H3K18ac. DIGIT is also enriched in regions of H3K18ac, and the depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings show that cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest that protein-lncRNA phase-separated condensates have a broader role as regulators of transcription.
| S-EPMC8008247 | biostudies-literature
Project description:Gene programs that control differentiation are regulated through the interplay between DNA, RNA, and protein. Cooperation among these fundamental cellular components can occur through highly structured interactions connecting domains formed by specific sequences of nucleotides, ribonucleotides, and/or amino acids and also through the assembly of biomolecular condensates. Here, we show that endoderm differentiation is regulated through the interaction of the long noncoding (lnc) RNA DIGITand the bromodomain and extra-terminal (BET) domain family protein BRD3. BRD3 forms phase-separated condensates that contain DIGIT, occupies enhancers of endoderm transcription factors, and is required for endoderm differentiation. Purified BRD3 binds to acetylated histone H3 lysine 18 (H3K18ac) in vitro and occupies regions of the genome enriched in H3K18ac during endoderm differentiation, including the key transcription factors that regulate endoderm differentiation. DIGIT is also enriched in regions of H3K18ac, and depletion of DIGIT results in decreased recruitment of BRD3 to these regions. Our findings support a model where cooperation between DIGIT and BRD3 at regions of H3K18ac regulates the transcription factors that drive endoderm differentiation and suggest a broader role for protein-lncRNA phase-separated condensates as regulators of transcription in development.
2020-07-08 | GSE126661 | GEO
Project description:Exosomes may mediate cell-to-cell communication by transporting various proteins and nucleic acids to neighboring cells. Some protein and RNA cargoes are significantly enriched in exosomes. How cells efficiently and selectively sort them into exosomes remains incompletely explored. Previously, we reported that YBX1 is required in sorting of miR-223 into exosomes. Here, we show that YBX1 undergoes liquid-liquid phase separation (LLPS) in vitro and in cells. YBX1 condensates selectively recruit miR-223 in vitro and into exosomes secreted by cultured cells. Point mutations that inhibit YBX1 phase separation impair the incorporation of YBX1 protein into biomolecular condensates formed in cells, and perturb miR-233 sorting into exosomes. We propose that phase separation-mediated local enrichment of cytosolic RNA-binding proteins and their cognate RNAs enables their targeting and packaging by vesicles that bud into multivesicular bodies. This provides a possible mechanism for efficient and selective engulfment of cytosolic proteins and RNAs into intraluminal vesicles which are then secreted as exosomes from cells.
| S-EPMC8612733 | biostudies-literature
Project description:Cells contain numerous membraneless organelles that assemble by intracellular liquid-liquid phase separation. The viscous properties and associated biomolecular mobility within these condensed phase droplets, or condensates, are increasingly recognized as important for cellular function and also dysfunction, for example, in protein aggregation pathologies. Fluorescence recovery after photobleaching (FRAP) is widely used to assess condensate fluidity and to estimate protein diffusion coefficients. However, the models and assumptions utilized in FRAP analysis of protein condensates are often not carefully considered. Here, we combine FRAP experiments on both in vitro reconstituted droplets and intracellular condensates with systematic examination of different models that can be used to fit the data and evaluate the impact of model choice on measured values. A key finding is that model boundary conditions can give rise to widely divergent measured values. This has important implications, for example, in experiments that bleach subregions versus the entire condensate, two commonly employed experimental approaches. We suggest guidelines for determining the appropriate modeling framework and highlight emerging questions about the molecular dynamics at the droplet interface. The ability to accurately determine biomolecular mobility both in the condensate interior and at the interface is important for obtaining quantitative insights into condensate function, a key area for future research.
| S-EPMC6818185 | biostudies-literature
Project description:Here, we provide evidence for the presence of Myosin phosphatase rho-interacting protein (MPRIP), an F-actin-binding protein, in the cell nucleus. The MPRIP protein binds to Phosphatidylinositol 4,5-bisphosphate (PIP2) and localizes to the nuclear speckles and nuclear lipid islets which are known to be involved in transcription. We identified MPRIP as a component of RNA Polymerase II/Nuclear Myosin 1 complex and showed that MPRIP forms phase-separated condensates which are able to bind nuclear F-actin fibers. Notably, the fibrous MPRIP preserves its liquid-like properties and reforms the spherical shaped condensates when F-actin is disassembled. Moreover, we show that the phase separation of MPRIP is driven by its long intrinsically disordered region at the C-terminus. We propose that the PIP2/MPRIP association might contribute to the regulation of RNAPII transcription via phase separation and nuclear actin polymerization.
| S-EPMC8068864 | biostudies-literature
Project description:Liquid-liquid phase separation of multivalent intrinsically disordered protein-RNA complexes is ubiquitous in both natural and biomimetic systems. So far, isotropic liquid droplets are the most commonly observed topology of RNA-protein condensates in experiments and simulations. Here, by systematically studying the phase behavior of RNA-protein complexes across varied mixture compositions, we report a hollow vesicle-like condensate phase of nucleoprotein assemblies that is distinct from RNA-protein droplets. We show that these vesicular condensates are stable at specific mixture compositions and concentration regimes within the phase diagram and are formed through the phase separation of anisotropic protein-RNA complexes. Similar to membranes composed of amphiphilic lipids, these nucleoprotein-RNA vesicular membranes exhibit local ordering, size-dependent permeability, and selective encapsulation capacity without sacrificing their dynamic formation and dissolution in response to physicochemical stimuli. Our findings suggest that protein-RNA complexes can robustly create lipid-free vesicle-like enclosures by phase separation.
| S-EPMC7354941 | biostudies-literature