RNA partitioning into stress granules is independent of G3BP
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE119977 | GEO | 2019/01/30
REPOSITORIES: GEO
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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.
2023-10-11 | GSE223295 | GEO
Project description:Stress granules (SGs) are dynamic, membrane-less organelles containing complex RNA-protein networks. The RNA components of SGs have been profiled through various approaches, characterized as long, translation-repressed, and highly epigenetically modified. However, it remains unclear whether these RNAs are uniformly distributed within SGs. In this study, we used multiple SG core proteins to genetically target the photocatalyst protein miniSOG, which allows for comprehensive CAP-seq profiling of RNA components within SGs. We discovered that RNAs associated with different SG core proteins exhibit heterogeneous distribution and distinct intrinsic features. Furthermore, we applied CAP-seq to map the RNA components in processing bodies (PBs) under both unstressed conditions and arsenite stimulation. By comparing the SG and PB transcriptomes, our data revealed that m6A modification may promote the localization of RNAs to SGs, while higher AU content may facilitate the targeting of mRNAs to PBs. This suggests potential regulatory roles in determining the subcellular localization of mRNAs within membrane-less organelles.
2024-09-29 | GSE271045 | GEO
Project description:Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress cells also limit protein synthesis by triggering transient storage of mRNAs and RNA binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated mutant of FUS in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus thereby linking the nuclear and cytosolic axis of proteotoxic stress response. Toidentify heat induced, RNF4 regulated ubiquitylation sites we performed an unbiased SILAC-based mass-spectrometry screen comparing heat-induced ubiquitylation in control and RNF4 depleted cells.
2023-06-30 | PXD017887 | Pride
Project description:Stress Granules (SG) formation is a cellular protection mechanism in harmful conditions, constituting a storage for untranslated mRNAs and RNA-binding proteins (RBPs); however, these condensates can turn into pathological aggregates, that in neurons are related to the onset of neurodegenerative diseases, like Amyotrophic Lateral Sclerosis (ALS). Mutations in the RBP FUS, leading to its cytoplasmic mis-localization, are causatively linked to ALS, since they mediate its accumulation in SGs, triggering their transition towards cytotoxic inclusions. Here, we describe the SG transcriptome in a neural context and compare with datasets from other systems, identifying both common rules and diversifying characteristics for SG recruitment of transcripts. We demonstrate that SG dynamics and RNA content are strongly modified by the incorporation of aberrantly localized mutant FUS, switching to a more unstructured, AU-rich SG transcriptome and losing a wide population of GC-rich, structured RNAs. We show that these alterations mainly depend on mutant FUS which favors the SG incorporation of its protein interactors and in turn of their target RNAs. Our data give a comprehensive view of the molecular differences between physiological and pathological SG in ALS conditions, showing how a single mutation is sufficient to impact the RNA and protein population of these condensates.
2024-12-06 | GSE244751 | GEO
Project description:Exposure of cells to heat or oxidative stress causes misfolding of proteins. To avoid toxic protein aggregation, cells have evolved nuclear and cytosolic protein quality control (PQC) systems. In response to proteotoxic stress cells also limit protein synthesis by triggering transient storage of mRNAs and RNA binding proteins (RBPs) in cytosolic stress granules (SGs). We demonstrate that the SUMO-targeted ubiquitin ligase (StUbL) pathway, which is part of the nuclear proteostasis network, regulates SG dynamics. We provide evidence that inactivation of SUMO deconjugases under proteotoxic stress initiates SUMO-primed, RNF4-dependent ubiquitylation of RBPs that typically condense into SGs. Impairment of SUMO-primed ubiquitylation drastically delays SG resolution upon stress release. Importantly, the StUbL system regulates compartmentalization of an amyotrophic lateral sclerosis (ALS)-associated mutant of FUS in SGs. We propose that the StUbL system functions as surveillance pathway for aggregation-prone RBPs in the nucleus thereby linking the nuclear and cytosolic axis of proteotoxic stress response. To better understand the role of RNF4 in the proteotoxic stress response we aimed to identify RNF4-associated protein complexes in cells exposed to heat stress. To allow immuno-affinity purification of endogenous RNF4 on anti-Flag agarose beads we used CRISPR/Cas9-mediated gene editing to integrate a C-terminal 3xFlag epitope tag into the genomic locus of RNF4 in HeLa cells. For the IP-MS experiment HeLa-RNF4-3xFlag cells or parental untagged control cells were exposed to heat stress for 1 h and RNF4-3xFlag together with associated proteins was captured on anti-Flag affinity beads. Tryptic peptides were analyzed by LC-MS/MS and data from three replicates were quantified by the Max Quant label-free quantification (LFQ) algorithm.
2023-06-30 | PXD017888 | Pride
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.
2023-02-03 | GSE220821 | GEO
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.
2019-01-07 | PXD011716 | Pride
Project description:Human salispheres, a culture of stem/progenitor cells, represent a potential therapy for radiation induced hyposalivation. Radiation-induced hyposalivation dramatically reduces quality of life of patients. We have demonstrated the potential of human salispheres to engraft and differentiate when transplanted into a mouse model of hyposalivation, in the manuscript associated with these data. We also demonstrate the functional recovery of irradiated salivary glands (SGs) following human salisphere transplantation, by the measurement of saliva production. When analyzing human salisphere- transplanted SGs for the presence of human cells, we noted that the highest proportion of human cells were present 1 week after transplantation. This microarray study was designed to determine if paracrine signaling from transplanted human salisphere cells to recipient mouse SGs accounts for a part of the functional recovery of the recipient SG we observe. We compare the transcriptome from irradiated control SG of immunecompromised NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice with that of NSG SGs transplanted with 100,000 human salisphere cells. All mice were sacrificed 1 week following transplantation (or non-transplantation in case of controls) and Illumina array chips used to detect differences in gene expression. 6 samples were analysed in total. Total RNA from 3 irradiated control SGs (5Gy irradiation) and 3 salivary glands transplanted with 100,000 human salispheres.
2015-09-10 | E-GEOD-72869 | biostudies-arrayexpress
Project description:Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into stress granules (SGs) reduces translation, but it remains unclear whether SGs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress-adaptation in cancer. Transcripts enriched in PSs, defined by polysome fractionation and RNAseq, were compared with mRNAs complexed with the SG-nucleator protein, G3BP1, defined by spatially-restricted enzymatic tagging. Short-term oxidative stress profoundly altered mRNA translation by promoting selective enrichment of transcripts within SGs or PSs. G3BP1 participates in this compartmentalisation by sequestering transcripts in SGs. Under stress, G3BP1-bound transcripts are PS-depleted and encode proteins involved in mRNA translation, pro-apoptosis, and mitochondrial function. In contrast, specific PS-enriched transcripts disassociate from G3BP1 under stress to encode proteins involved in diverse cellular cytoprotective pathways. Therefore, G3BP1 partitioning guides selective translation to support stress adaptation and cell survival.
2020-04-30 | PXD015491 | Pride
Project description:Cells limit energy-consuming mRNA translation during stress to maintain metabolic homeostasis. Sequestration of mRNAs by RNA binding proteins (RBPs) into stress granules (SGs) reduces translation, but it remains unclear whether SGs also function in partitioning of specific transcripts to polysomes (PSs) to guide selective translation and stress-adaptation in cancer. Transcripts enriched in PSs, defined by polysome fractionation and RNAseq, were compared with mRNAs complexed with the SG-nucleator protein, G3BP1, defined by spatially-restricted enzymatic tagging. Short-term oxidative stress profoundly altered mRNA translation by promoting selective enrichment of transcripts within SGs or PSs. G3BP1 participates in this compartmentalisation by sequestering transcripts in SGs. Under stress, G3BP1-bound transcripts are PS-depleted and encode proteins involved in mRNA translation, pro-apoptosis, and mitochondrial function. In contrast, specific PS-enriched transcripts disassociate from G3BP1 under stress to encode proteins involved in diverse cellular cytoprotective pathways. Therefore, G3BP1 partitioning guides selective translation to support stress adaptation and cell survival.
2019-11-01 | GSE138058 | GEO