Isolation of yeast and mammalian stress granule cores

Wheeler, Joshua; Jain, Shuchi; Khong, Anthony; Parker, Roy

doi:10.1016/j.ymeth.2017.04.020

Cited by 101 publications

(96 citation statements)

References 13 publications

(24 reference statements)

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“…To this end, BV2 GFP-G3BP1 cells were either treated with 0.1 mM arsenite for 1h to induce SG assembly or infected with MNV for 9h. G3BP1 granules and SG cores were then enriched by sequential centrifugation and purified by immunoprecipitation using antibodies to GFP (to trap GFP-G3BP1) or IgG (as a control) followed by pull down with Protein A-conjugated Dynabeads as previously described (68). Epifluorescence microscopy analysis then confirmed the isolation on anti-GFP beads of SG core or G3BP1 granules, while no granules could be detected in the control IgG immunoprecipitation ( Figure 5A).…”

Section: Mnv-induced G3bp1 Granules Differ From Canonical Sgmentioning

confidence: 64%

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

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Iadevaia

Klein

et al. 2018

Preprint

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During viral infection, the accumulation of RNA replication intermediates or viral proteins imposes major stress on the host cell. In response, cellular stress pathways can rapidly impose defence mechanisms by shutting off the protein synthesis machinery, which viruses depend on, and triggering the accumulation of mRNAs into stress granules to limit the use of energy and nutrients. Because this threatens viral gene expression, viruses need to evade these pathways to propagate. Human norovirus is responsible for gastroenteritis outbreaks worldwide. Previously we showed that murine norovirus (MNV) regulates the activity of eukaryotic initiation factors (eIFs). Here we examined how MNV interacts with the eIF2a signaling axis controlling translation and stress granules accumulation.We show that while MNV infection represses host cell translation, it results in the assembly of virusspecific granules rather than stress granules. Further mechanistic analyses revealed that eIF2a signaling is uncoupled from translational stalling. Moreover the interaction of the RNA-binding protein G3BP1 with viral factors together with a redistribution of its cellular interacting partners could explain norovirus evasion of stress granules assembly. These results identify novel strategies by which norovirus ensure efficient replication propagation by manipulating the host stress response.

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Section: Mnv-induced G3bp1 Granules Differ From Canonical Sgmentioning

confidence: 64%

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

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Iadevaia

Klein

et al. 2018

Preprint

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“…P body-enriched yeast cell lysate was prepared in a manner similar to the stress granule enrichment described previously (18,36). Briefly, frozen yeast cell pellets resuspended in 1× lysis buffer were disrupted using four 10-min rounds of glass bead beating with intermittent cooling.…”

Section: Methodsmentioning

confidence: 99%

Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae

Rao

Parker

2017

Proc. Natl. Acad. Sci. U.S.A.

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108

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Eukaryotic cells contain multiple RNA-protein assemblies referred to as RNP granules, which are thought to form through multiple protein-protein interactions analogous to a liquid-liquid phase separation. One class of RNP granules consists of P bodies, which consist of nontranslating mRNAs and the general translation repression and mRNA degradation machinery. P bodies have been suggested to form predominantly through interactions of Edc3 and a prion-like domain on Lsm4. In this work, we provide evidence that P-body assembly can be driven by multiple different protein-protein and/or protein-RNA interactions, including interactions involving Dhh1, Psp2, and Pby1. Moreover, the relative importance of specific interactions can vary with different growth conditions. Based on these observations, we develop a summative model wherein the P-body assembly phenotype of a given mutant can be predicted from the number of currently known protein-protein interactions between P-body components.

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“…SGs are composed of stable cores and a more dynamic and fluid shell. Excitingly, the SG cores have recently been isolated from mammalian and yeast cells using a combination of a differential centrifugation steps and AP (Jain et al, 2016;Wheeler et al, 2017). We decided to adapt this existing protocol to plant cells to investigate the composition of SGs in plants (Fig.…”

Section: Sg Cores Can Be Isolated From Plant Materialsmentioning

confidence: 99%

Protein and metabolite composition of Arabidopsis stress granules

et al. 2019

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Summary 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. In this study, we addressed the existing gap. Importantly, we also provide evidence for metabolite sequestration within the SGs. To isolate SGs we used Arabidopsis seedlings expressing green fluorescent protein (GFP) fusion of the SGs marker protein, Rbp47b, and an experimental protocol combining differential centrifugation with affinity purification (AP). SGs isolates were analysed 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, nucleotides, amino acids and phospholipids also accumulated in SGs. Taken together, our results indicated the presence of a preexisting SG protein interaction network; an evolutionary conservation of the proteins involved in SG assembly and dynamics; an important role for SGs in moderation of stress responses by selective storage of proteins and metabolites.

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Isolation of yeast and mammalian stress granule cores

Cited by 101 publications

References 13 publications

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

Norovirus infection results in assembly of virus-specific G3BP1 granules and evasion of eIF2α signaling

Numerous interactions act redundantly to assemble a tunable size of P bodies in Saccharomyces cerevisiae

Protein and metabolite composition of Arabidopsis stress granules

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