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The adaptor protein AP-3β disassembles heat-induced stress granules via 19S RP-associated deubiquitylation in Arabidopsis

ABSTRACT: To survive under adverse conditions, plants form stress granules (SGs) to temporally store mRNA and halt translation as a primary response. Dysregulation in SG disassembly can have detrimental effects on plant survival after stress release, yet the underlying mechanism remains poorly understood. Using Arabidopsis as a model system, we demonstrated that the AP-3 subunit AP-3β participates in heat response independently of its conventional role in vacuolar transportinteracts with the SG core RNA-binding proteins TSN1/2 in vivo and in vitro. We also showed that AP-3β is rapidly recruited to SGs upon heat induction and plays a key role in SG disassembly after heat release. We also discovered thatGenetic evidences support that AP-3β serves as an adaptor to recruit the 19S regulatory particle (RP) of the proteasome to SGs upon heat induction. Notably, the 19S RP promotes SG disassembly through RP-associated deubiquitylation, independent of its proteolytic catalytic activity. This deubiquitylation process of SG components is crucial for translation reinitiation and growth recovery after heat release. Our findings shed light on the non-proteolytic catalytic function of the RP in regulating SG dynamics disassembly and reveal highlight the importance of endomembrane proteins in supporting RNA granule dynamics in plant cells.a non-degradation mechanism for cellular adaptation to environmental stresses.

INSTRUMENT(S): timsTOF Pro

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Plant Cell, Seedling

SUBMITTER: Lei Pang

LAB HEAD: Ruixi Li

PROVIDER: PXD058194 | Pride | 2025-01-24

REPOSITORIES: pride

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	Ub-HS-Col-0_1_28089.d.zip	Other
	Ub-HS-Col-0_2_21791.d.zip	Other
	Ub-HS-Col-0_3_21793.d.zip	Other

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The 19S RP-associated deubiquitylation downstream of AP-3β disassembles heat-induced stress granules in plants

Project description:To survive under adverse conditions, plants form stress granules (SGs) to temporally store mRNA and halt translation as a primary response. Dysregulation in SG disassembly can have detrimental effects on plant survival after stress release, yet the underlying mechanism remains poorly understood in plants. Using Arabidopsis as a model system, we demonstrated that the AP-3 subunit AP-3β participates in heat response independently of its conventional role in vacuolar transport. We also discovered that AP-3β serves as an adaptor to recruit the 19S regulatory particle (RP) of the proteasome to SGs upon heat induction. Notably, the 19S RP promotes SG disassembly through RP-associated deubiquitylation, independent of its proteolytic activity. This deubiquitylation process of SG components is crucial for translation reinitiation and growth recovery after heat release. Our findings shed light on the non-proteolytic function of the 19S RP in regulating SGs dynamics and provide insights into a non-degradation mechanism for cellular adaptation to environmental stresses

2025-01-24 | PXD054299 | Pride

Proteasome resides in and dismantles plant heat stress granules constitutively

Project description:Stress granules (SGs) are conserved reversible cytoplasmic condensates enriched with aggregation-prone proteins assembled in response to various stresses. Defects in disassembly have been associated with various neuro- and muscular degenerative diseases in humans. Plants appear to have efficient approaches to avoid the pathological conversion of SGs. However, how plants regulate SG dynamics is unclear. Here we show increase in either temperature or duration of heat stress reduces the molecular mobility of SG marker protein and suppresses SG clearance. Proteomics analysis and FRAP assays demonstrated 20S and 26S proteasomes as stable “core” components of SGs recruited early during the heat stress. Strikingly, while heat stress inhibits the activity of the overall proteasomes, it induces dramatic ubiquitylation of SG components and enhances the activities of SG-resident proteasomes, which degrade SG components even during the assembly phase. Their proteolytic activities enable the timely disassembly of SGs and secure the survival of plant cells during the recovery from heat stress.

2024-08-22 | PXD044922 | Pride

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.

2019-01-07 | PXD011716 | Pride

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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The Small Molecule ISRIB Reverses the Effects of eIF2α Phosphorylation on Translation and Stress Granule Assembly

Project description:We recently identified ISRIB as a potent inhibitor of the integrated stress response (ISR). ISRIB renders cells resistant to the effects of eIF2α phosphorylation and enhances long-term memory in rodents (10.7554/eLife.00498). Here we show by genome-wide in vivo ribosome profiling that translation of a restricted subset of mRNAs is induced upon ISR activation. ISRIB substantially reversed the translational effects elicited by phosphorylation of eIF2α and induced no major changes in translation or mRNA levels in unstressed cells. eIF2α phosphorylation-induced stress granule (SG) formation was blocked by ISRIB. Strikingly, ISRIB addition to stressed cells with pre-formed SGs induced their rapid disassembly, liberating mRNAs into the actively translating pool. Restoration of mRNA translation and modulation of SG dynamics may be an effective treatment of neurodegenerative diseases characterized by eIF2α phosphorylation, SG formation and cognitive loss. Ribosome profiling with paired RNA-seq

2015-02-27 | E-GEOD-65778 | biostudies-arrayexpress

Developmental profiling of spiral ganglion neurons reveals insights into auditory circuit assembly

Project description:The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons at six developmental stages, ranging from embryonic day 12 (E12), when SG neurons first extend projections, up until postnatal day 15 (P15), after the onset of hearing. For comparison, we also analyzed the closely-related vestibular ganglion (VG) at the same time points. To identify genes involved in SG axon guidance and branching, target selection, synaptogenesis, synaptic refinement, and synaptic function, we collected SG at E12 and E13, E16, P0, P6, and P15. We also collected VG at the same time points. For E12 and E13 time points, SG and VG were microdissected from Rnx-cre; Z/EG embryos, which express GFP in the VG. E16-P15 VG was also isolated by microdissection from Rnx-cre; Z/EG animals. E16-P15 SG neurons were isolated by FACS sorting dissociated cochlea from Mafb-GFP animals.

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Reversible amyloids of pyruvate kinase couple cell metabolism and stress granule disassembly

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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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