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Stress granule phase separation in stress-responsive cytosolic extract-in-oil droplets

ABSTRACT: Stress granules (SGs) are biomolecular condensates that form transiently in the cytosol of mammalian cells in response to stress. Dysregulation of their assembly or disassembly is implicated in human age-related diseases. While phase separation is the key process underlying SG assembly, understanding of their function, composition and regulation in response to physiological stimuli is limited. This knowledge gap reflects the challenge of gaining comprehensive and quantitative insights into the dynamic regulation of the complex composition of SGs at the single-cell level. Here we present an emulsion-based microfluidics method to overcome this limitation. "Cytosolic extracts-in-oil droplets” (CEODs) recreate a confined active cytosolic milieu that undergoes phase separation and SG formation in response to stress under physiological conditions. This approach led to the discovery of six novel SG components involved in signalling pathways. CEODs provide a versatile and cost-effective screening platform for future mechanistic and therapeutic studies.

ORGANISM(S): Homo sapiens

PROVIDER: GSE308184 | GEO | 2026/04/24

REPOSITORIES: GEO

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RNA partitioning into stress granules is independent of G3BP

Project description:Stress granules (SGs) are stress induced RNA-protein assemblies formed from untranslating mRNPs. Mammalian SGs are non-uniform and contain “cores”, which are stable in lysates and heterogenous in protein composition. The interactions defining RNA recruitment into SGs, and whether the RNA composition varies between different cores remains unclear. Herein, we determine the SG transcriptome through purification of both PABPC1 and G3BP SG cores during both arsenite and sorbitol stress. We observe that similar RNAs are recruited to SGs independent of the protein used for core purification, suggesting individual RNA-binding proteins (RBPs) may play a limited role in defining the SG transcriptome. Analysis of the sorbitol-induced SG transcriptome reveals G3BP1/2 has little effect on RNAs recruited to SGs suggesting RNAs localize to SGs independent of individual RBPs. Taken together, we suggest that partitioning of RNAs to SGs is independent of at least some proteins, consistent with SGs forming through intermolecular RNA-RNA interactions.

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Profiling stress-triggered RNA condensation with photocatalytic proximity labeling

Project description:Stress granules (SGs) are highly dynamic cytoplasmic membrane-less organelles that assemble when cells are challenged by stress. At different stages of assembly, various RNA molecules are sorted into SGs where they play important roles in maintaining the structural stability of SGs and regulating gene expression. Despite recent efforts of fluorescence microscopy and biochemical fractionation analysis, there still lacks a complete description of the dynamic changes in the molecular inventory of SG components during different stages of assembly and disassembly. In this study, we applied a proximity-dependent RNA labeling method, CAP-seq, to comprehensively investigate the content of local transcriptome in SGs, in the context of live mammalian cells. CAP-seq captures 457 and 822 RNAs in arsenite- and sorbitol-induced SGs in HEK293T cells, respectively, revealing that SG enrichment is positively correlated with RNA length and AU content, but negatively correlated with translation efficiency. The high spatial specificity of CAP-seq dataset is validated by single-molecule FISH imaging. We further applied CAP-seq profile RNA components in microscopically invisible SG cores, both before stress and after recovery from stress, thus mapping the dynamic changes in SG-proximal transcriptome along the time course of granule assembly/disassembly processes. Our data portray a model of AU-rich and translationally repressed SG nanostructure that are memorized long after the removal of stress.

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Protein and metabolite composition of Arabidopsis stress granules

Project description:• Stress granules (SGs) are evolutionary conserved aggregates of proteins and untranslated mRNAs formed in response to stress. Despite their importance for stress adaptation, no complete proteome composition has been reported for plant SGs. Herein, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs. • To isolate SGs we used Arabidopsis seedlings expressing GFP fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification. SGs isolates were analyzed using mass spectrometry-based proteomics and metabolomics. • A quarter of the identified proteins constituted known or predicted SG components. Intriguingly, the remaining proteins were enriched in key enzymes and regulators, such as cyclin dependent kinase A (CDKA), that mediate plant responses to stress. In addition to proteins, also nucleotides, amino acids and phospholipids accumulated in SGs. • Taken together, our results indicate (i) the presence of a pre-existing SG protein interaction network, (ii) an evolutionary conservation of the proteins involved in SG assembly and dynamics, (iii) an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.

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Diverse CMT2 Neuropathies are Linked to Aberrant G3BP Interactions in Stress Granules

Project description:The formation of stress granules (SGs) is an important anti-stress mechanism in response to environmental insults. SG is a type of discrete cytoplasmic foci composed of translationally silent ribonucleoproteins. To explore the composition of SGs, we use CLIP-Seq to detect the RNAs associated with G3BP1.

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Revealing Stress Granule Compositional Heterogeneity through Antibody-Guided Proximity Labeling

Project description:Stress granules (SGs), transient nonmembranous cytoplasmic condensates that formed in response to cellular stresses, require precise characterization to unravel their cell-type and stressspecific protein compositions. This study introduced a G3BP1 antibody-guided proximity labeling (Ab-PL) method to explore the composition and diversity of SGs, overcoming the challenges of traditional enzyme-mediated proximity labeling techniques across various cell types, especially for the immune cells. Application of Ab- PL to HeLa and RAW264.7 cells under heat shock (HS), sodium arsenate (AS), and sodium chloride stress (SS) revealed two categories of SG proteins: “SG-core” and “SG-shell,” characterized by their different abilities to undergo phase separation. The core proteins form the SG scaffold with strong self-segregation, while shell proteins are dynamically recruited based on the type of stress. Cell- and stress-specific SG proteins were also identified, highlighting compositional heterogeneity. Intriguingly, unique nuclear-cytoplasmic shuttling behaviors of SG components were observed under varying conditions, uncovering over 10 novel SG proteins, including REXO4, RBM28, and OGFR. This study provides a versatile tool for SG analysis across diverse cell types and offers insights into SG heterogeneity, which has potential implications for human diseases, paving the way for future studies on RNA metabolism, ribosome assembly, and immune regulation.

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