Synthetic biomolecular condensates enhance translation from a target mRNA in living cells
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PROVIDER: PRJNA1162121 | ENA |
REPOSITORIES: ENA
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Project description:Biomolecular condensates composed of proteins and RNA are one approach by which cells regulate post-transcriptional gene expression. Their formation typically involves the phase separation of intrinsically disordered proteins with a target mRNA, sequestering the mRNA into a liquid condensate. This sequestration regulates gene expression by modulating translation or facilitating RNA processing. Here, we engineer synthetic condensates using a fusion of an RNA-binding protein, the human Pumilio2 homology domain, and a synthetic intrinsically disordered protein, an elastin-like polypeptide, that can bind and sequester a target mRNA transcript. In protocells, sequestration of a target mRNA largely limits its translation. Conversely, in E. Coli, sequestration of the same target mRNA increases its translation. We characterize the Pum2-ELP condensate system using microscopy, biophysical and biochemical assays, and RNA-seq. This approach enables modulation of cell function via the formation of synthetic biomolecular condensates that upregulate the expression of a target protein.
2024-11-30 | GSE277409 | GEO
Project description:Biomolecular condensates play important roles in diverse biological processes. Many viruses form biomolecular condensates which have been implicated in various functions critical for productive infection of host cells. The adenovirus L1-52/55 kilodalton protein (52K) was recently shown to form viral biomolecular condensates that coordinate viral genome packaging and capsid assembly. Although critical for packaging, we do not know how viral condensates are regulated during adenovirus infection. Here we show that phosphorylation of serine residues 28 and 75 within the N-terminal intrinsically disordered region of 52K modulates viral condensates in vitro and in cells, promoting liquid-like properties over condensate hardening. Furthermore, we demonstrate that phosphorylation of 52K promotes viral genome packaging and production of infectious progeny particles. Collectively, our findings provide insights into how viral condensate properties are regulated and maintained in a state conducive to their function in viral progeny production. In addition, our findings have implications for antiviral strategies aimed at targeting the regulation of viral biomolecular condensates to limit viral multiplication.
2023-12-06 | PXD046971 | Pride
Project description:It remains largely unclear how material properties of biomolecular condensates impact physiological and pathological processes such as cancer. Here we show that AKAP95, a nuclear protein involved in gene regulation, plays an important role in tumorigenesis through directly regulating mRNA splicing. We show that AKAP95, through an intrinsic disordered region (IDR), forms liquid-like droplets in vitro and dynamic foci in nucleus. We have also identified key residues in IDR whose mutations to different residues perturb AKAP95 condensation in opposite directions. Importantly, the biological activity of AKAP95 in splice regulation is affected by either disruption of liquid condensation or enhancing the gelation of the assembly. We then show that the ability of AKAP95 in overcoming oncogene-induced senescence requires AKAP95 in an appropriate window of liquidity. These results thus link phase separation to tumorigenesis and uncover an important role of regulating the material property of protein condensates in disease including cancer.
2020-05-06 | GSE122308 | GEO
Project description:Phase separation inside mammalian cells regulates the formation of biomolecular condensates that are related to gene expression, signalling, development, and diseases. However, a large population of endogenous condensates and their candidate phase separating proteins have yet to be discovered in a quantitative and high-throughput manner. Here, we demonstrate that endogenously-expressed biomolecular condensates can be identified across a cell's proteome by sorting proteins across varying oligomeric states. We employ volumetric compression to modulate the concentrations of intracellular proteins and the degree of crowdedness, which are physical regulators of cellular biomolecular condensates. The changes in degree of the partition of proteins into condensates or phase separation lead to varying oligomeric states of the proteins, which can be detected by coupling density gradient ultracentrifugation and quantitative mass spectrometry. In total, we identified 1,518 endogenous-expression condensate proteins, of which 538 have not been reported before. Furthermore, we demonstrate that our strategy can identify condensate proteins that respond to specific biological processes.
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Project description:Material properties of biomolecular condensates regulates RNA splicing and tumorigenesis
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Project description:Compartmentalization is an essential feature of eukaryotic life and is achieved both via membrane-bound organelles, such as mitochondria, and membrane-less biomolecular condensates, such as the nucleolus. Known biomolecular condensates typically exhibit liquid-like properties and are visualized by microscopy on the scale of ~1µm. They have been studied mostly by microscopy, examining select individual proteins. So far, several dozen biomolecular condensates are known, serving a multitude of functions, for example, in the regulation of transcription, RNA processing or signalling and their malfunction can cause diseases. However, it remains unclear to what extent biomolecular condensates are utilized in cellular organization and at what length scale they typically form. Here we examine native cytoplasm from Xenopus egg extract on a global scale with quantitative proteomics, filtration, size exclusion and dilution experiments. These assays reveal that at least 18% of the proteome is organized into mesoscale biomolecular condensates at the scale of ~100nm and appear to be stabilized by RNA or gelation. We confirmed mesoscale sizes via imaging below the diffraction limit by investigating protein permeation into porous substrates with defined pore sizes. Our results show that eukaryotic cytoplasm organizes extensively via biomolecular condensates, but at surprisingly short length scales.
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2024-10-09 | PXD042544 | Pride
Project description:Isolation and characterization of Dact1 biomolecular condensates with and without Wnt stimulation in breast cancer bone metastatic cells.
2021-01-18 | PXD019271 | Pride
Project description:While eukaryotic cells have a myriad of membrane-bound organelles enabling the isolation of different chemical environments, prokaryotic cells lack these defined reaction vessels. Biomolecular condensates—organelles that lack a membrane—provide a strategy for cellular organization without a physical barrier while allowing for the dynamic, responsive organization of the cell. It is well established that intrinsically disordered protein domains drive condensate formation via liquid–liquid phase separation; however, the role of globular protein domains on intracellular phase separation remains poorly understood. We hypothesized that the overall charge of globular proteins would dictate the formation and concentration of condensates and systematically probed this hypothesis with supercharged proteins and nucleic acids in E. coli. Within this study, we demonstrated that condensates form via electrostatic interactions between engineered proteins and RNA and that these condensates are dynamic and only enrich specific nucleic acid and protein components. Herein, we propose a simple model for the phase separation based on protein charge that can be used to predict intracellular condensate formation. With these guidelines, we have paved the way to designer functional synthetic membraneless organelles with tunable control over globular protein function.
2020-04-24 | MSV000085333 | MassIVE
Project description:Intracellular aggregation of repeat expanded RNA has been implicated in many neurological disorders. Here, we study the role of biomolecular condensates on irreversible RNA clustering. We find that physiologically relevant and disease-associated repeat RNAs spontaneously undergo an age-dependent percolation transition inside multi-component protein-nucleic acid condensates to form nanoscale clusters. Homotypic RNA clusters drive the emergence of multiphasic condensate structures with an RNA-rich solid core surrounded by an RNA-depleted fluid shell. The timescale of the RNA clustering, which drives a liquid-to-solid transition of biomolecular condensates, is determined by the sequence features, stability of RNA secondary structure, and repeat length. Importantly, G3BP1, the core scaffold of stress granules, introduces heterotypic buffering to homotypic RNA-RNA interactions and impedes intra-condensate RNA clustering in an ATP-independent manner. Our work suggests that biomolecular condensates can act as sites for RNA aggregation. It also highlights the functional role of RNA-binding proteins in suppressing aberrant RNA phase transitions.
| S-EPMC11195159 | biostudies-literature