Synthetic biomolecular condensates enhance translation from a target mRNA in living cells
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ABSTRACT:
PROVIDER: PRJNA1162121 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
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| SRR30693135_1.fastq.gz | Fastqsanger.gz | |||
| SRR30693135_2.fastq.gz | Fastqsanger.gz | |||
| SRR30693136_1.fastq.gz | Fastqsanger.gz | |||
| SRR30693136_2.fastq.gz | Fastqsanger.gz | |||
| SRR30693137_1.fastq.gz | Fastqsanger.gz |
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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.
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Project description:Biomolecular condensates are involved in a variety of neurodegenerations. These condensates are formed through phase separation mechanism. However, how they may be functional in the pathophysiology of Alzheimer’s disease (AD) remains poorly understood. Here, we report that in the brain of AD patients and animal models, an elevation of poly(C)-binding protein 2 (PCBP2) correlates with biomolecular condensation that involves phase separation. These condensates sequester large numbers of mitochondrial and mRNA-binding proteins, leading to the outside impairment of mitochondrial morphology and function, and BACE1 mRNA decay relative to amyloid deposition. We then identify a small molecule CN-0928 that inhibits the condensates by reducing PCBP2 protein level and mitigates AD pathology and cognitive decline, in which CN-0928 binding to a target protein integrator complex subunit 1 (INTS1) allows to regulate PCBP2 expression. Our findings place PCBP2 condensates as a key player that cooperates the seemingly disparate but important pathways, and show pharmacological modulation of PCBP2 as an effective approach for treating AD.
2025-09-30 | GSE306042 | GEO
Project description:Recent studies have emphasized the significance of biomolecular condensates in modulating gene expression through RNA processing and translational control. However, the functional roles of RNA condensates in cell fate specification remains poorly understood. Here, we profiled the coding and non-coding transcriptome within intact biomolecular condensates, specifically P-bodies, in diverse developmental contexts, spanning multiple vertebrate species. Our analyses revealed the conserved, cell type-specific sequestration of untranslated RNAs encoding key cell fate regulators. Notably, P-body contents did not directly reflect active gene expression profiles for a given cell type, but rather were enriched for translationally repressed transcripts characteristic of the preceding developmental stage. Mechanistically, microRNAs (miRNAs) direct the selective sequestration of RNAs into P-bodies in a context-dependent manner, and perturbing AGO2 or alternative polyadenylation profoundly reshapes P-body RNA content. Building on these mechanistic insights, we demonstrate that modulating P-body assembly or miRNA activity dramatically enhances both activation of a totipotency transcriptional program in naïve pluripotent stem cells as well as the programming of primed human embryonic cells towards the germ cell lineage. Collectively, our findings establish a direct link between biomolecular condensates and cell fate decisions across vertebrate species and provide a novel framework for harnessing condensate biology to expand clinically relevant cell populations.
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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.
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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: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.
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Project description:Material properties of biomolecular condensates regulates RNA splicing and tumorigenesis
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Project description:Selective RNA sequestration in biomolecular condensates directs cell fate transitions
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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