scholarly journals The catalytic activity of the Ubp3 deubiquitinating protease is required for efficient stress granule assembly inS. cerevisiae

2015 ◽  
pp. MCB.00609-15 ◽  
Author(s):  
Regina Nostramo ◽  
Sapna N. Varia ◽  
Bo Zhang ◽  
Megan M. Emerson ◽  
Paul K. Herman
Keyword(s):  
Catalytic Activity ◽  
Stress Granules ◽  
Eukaryotic Cell ◽  
Stress Granule ◽  
Resting Cells ◽  
Term Survival ◽  
General Role ◽  
Rnp Complex ◽  
Processing Body ◽  
Rnp Granules

The interior of the eukaryotic cell is a highly compartmentalized space containing both membrane-bound organelles and the recently-identified nonmembranous ribonucleoprotein (RNP) granules. This study examines inSaccharomyces cerevisiaethe assembly of one conserved type of the latter compartment, known as the stress granule. Stress granules form in response to particular environmental cues and have been linked to a variety of human diseases, including amyotrophic lateral sclerosis. To further our understanding of these structures, a candidate genetic screen was employed to identify regulators of stress granule assembly in quiescent cells. These studies identified a ubiquitin-specific protease, Ubp3, as having an essential role in the assembly of these RNP granules. This function was not shared by other members of the Ubp protease family and required Ubp3 catalytic activity as well as its interaction with the cofactor, Bre5. Interestingly, the loss of stress granules was correlated with a decrease in the long-term survival of stationary phase cells. This phenotype is similar to that observed in mutants defective for the formation of a related RNP complex, the Processing-body. Altogether, these observations raise the interesting possibility of a general role for these types of cytoplasmic RNP granules in the survival of G0-like resting cells.

scholarly journals Transcriptome-Wide Comparison of Stress Granules and P-Bodies Reveals that Translation Plays a Major Role in RNA Partitioning

2019 ◽  
Vol 39 (24) ◽  
Author(s):  
Tyler Matheny ◽  
Bhalchandra S. Rao ◽  
Roy Parker
Keyword(s):  
Stress Granules ◽  
Stress Granule ◽  
Dominant Factor ◽  
Differential Centrifugation ◽  
P Bodies ◽  
Translation Repression ◽  
First Approximation ◽  
Rnp Granules ◽  
Interpretation Of Results

ABSTRACT The eukaryotic cytosol contains multiple RNP granules, including P-bodies and stress granules. Three different methods have been used to describe the transcriptome of stress granules or P-bodies, but how these methods compare and how RNA partitioning occurs between P-bodies and stress granules have not been addressed. Here, we compare the analysis of the stress granule transcriptome based on differential centrifugation with and without subsequent stress granule immunopurification. We find that while differential centrifugation alone gives a first approximation of the stress granule transcriptome, this methodology contains nonspecific transcripts that play a confounding role in the interpretation of results. We also immunopurify and compare the RNAs in stress granules and P-bodies under arsenite stress and compare those results to those for the P-body transcriptome described under nonstress conditions. We find that the P-body transcriptome is dominated by poorly translated mRNAs under nonstress conditions, but during arsenite stress, when translation is globally repressed, the P-body transcriptome is very similar to the stress granule transcriptome. This suggests that translation is a dominant factor in targeting mRNAs into both P-bodies and stress granules, and during stress, when most mRNAs are untranslated, the composition of P-bodies reflects this broader translation repression.

scholarly journals Puumala and Andes Orthohantaviruses Cause Transient Protein Kinase R-Dependent Formation of Stress Granules

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Wanda Christ ◽  
Janne Tynell ◽  
Jonas Klingström
Keyword(s):  
Protein Kinase ◽  
Host Cell ◽  
Stress Responses ◽  
Stress Granules ◽  
Cell Stress ◽  
Stress Granule ◽  
Hantavirus Infection ◽  
Stress Signaling ◽  
Protein Kinase R ◽  
Granule Formation

ABSTRACT Virus infection frequently triggers host cell stress signaling resulting in translational arrest; as a consequence, many viruses employ means to modulate the host stress response. Hantaviruses are negative-sense, single-stranded RNA viruses known to inhibit host innate immune responses and apoptosis, but their impact on host cell stress signaling remains largely unknown. In this study, we investigated activation of host cell stress responses during hantavirus infection. We show that hantavirus infection causes transient formation of stress granules (SGs) but does so in only a limited proportion of infected cells. Our data indicate some cell type-specific and hantavirus species-specific variability in SG prevalence and show SG formation to be dependent on the activation of protein kinase R (PKR). Hantavirus infection inhibited PKR-dependent SG formation, which could account for the transient nature and low prevalence of SG formation observed during hantavirus infection. In addition, we report only limited colocalization of hantaviral proteins or RNA with SGs and show evidence indicating hantavirus-mediated inhibition of PKR-like endoplasmic reticulum (ER) kinase (PERK). IMPORTANCE Our work presents the first report on stress granule formation during hantavirus infection. We show that hantavirus infection actively inhibits stress granule formation, thereby escaping the detrimental effects on global translation imposed by host stress signaling. Our results highlight a previously uncharacterized aspect of hantavirus-host interactions with possible implications for how hantaviruses are able to cause persistent infection in natural hosts and for pathogenesis.

scholarly journals Translationally Repressed mRNA Transiently Cycles through Stress Granules during Stress

2008 ◽  
Vol 19 (10) ◽  
pp. 4469-4479 ◽  
Author(s):  
Stephanie Mollet ◽  
Nicolas Cougot ◽  
Ania Wilczynska ◽  
François Dautry ◽  
Michel Kress ◽  
...  
Keyword(s):  
Residence Time ◽  
Kinetic Data ◽  
Stress Granules ◽  
Stress Relief ◽  
Mrna Degradation ◽  
Stress Granule ◽  
Storage Site ◽  
Direct Role ◽  
Close Proximity

In mammals, repression of translation during stress is associated with the assembly of stress granules in the cytoplasm, which contain a fraction of arrested mRNA and have been proposed to play a role in their storage. Because physical contacts are seen with GW bodies, which contain the mRNA degradation machinery, stress granules could also target arrested mRNA to degradation. Here we show that contacts between stress granules and GW bodies appear during stress-granule assembly and not after a movement of the two preassembled structures. Despite this close proximity, the GW body proteins, which in some conditions relocalize in stress granules, come from cytosol rather than from adjacent GW bodies. It was previously reported that several proteins actively traffic in and out of stress granules. Here we investigated the behavior of mRNAs. Their residence time in stress granules is brief, on the order of a minute, although stress granules persist over a few hours after stress relief. This short transit reflects rapid return to cytosol, rather than transfer to GW bodies for degradation. Accordingly, most arrested mRNAs are located outside stress granules. Overall, these kinetic data do not support a direct role of stress granules neither as storage site nor as intermediate location before degradation.

scholarly journals The Stress Granule Protein G3BP1 Recruits Protein Kinase R To Promote Multiple Innate Immune Antiviral Responses

2014 ◽  
Vol 89 (5) ◽  
pp. 2575-2589 ◽  
Author(s):  
Lucas C. Reineke ◽  
Richard E. Lloyd
Keyword(s):  
Protein Kinase ◽  
Antiviral Activity ◽  
Cellular Stress ◽  
Stress Granules ◽  
Innate Immune ◽  
Stress Granule ◽  
Productive Infection ◽  
Antiviral Protein ◽  
Protein Kinase R ◽  
Transcriptional Responses

ABSTRACTStress granules (SGs) are cytoplasmic storage sites containing translationally silenced mRNPs that can be released to resume translation after stress subsides. We previously showed that poliovirus 3C proteinase cleaves the SG-nucleating protein G3BP1, blocking the ability of cells to form SGs late in infection. Many other viruses also target G3BP1 and inhibit SG formation, but the reasons why these functions evolved are unclear. Previously, we also showed a link between G3BP1-induced SGs and protein kinase R (PKR)-mediated translational control, but the mechanism of PKR interplay with SG and the antiviral consequences are unknown. Here, we show that G3BP1 exhibits antiviral activity against several enteroviruses, whereas truncated G3BP1 that cannot form SGs does not. G3BP1-induced SGs are linked to activation of innate immune transcriptional responses through NF-κB and JNK. The G3BP1-induced SGs also recruit PKR and other antiviral proteins. We show that the PXXP domain within G3BP1 is essential for the recruitment of PKR to SGs, for eIF2α phosphorylation driven by PKR, and for nucleating SGs of normal composition. We also show that deletion of the PXXP domain in G3BP1 compromises its antiviral activity. These findings tie PKR activation to its recruitment to SGs by G3BP1 and indicate that G3BP1 promotes innate immune responses at both the transcriptional and translational levels and integrates cellular stress responses and innate immunity.IMPORTANCEStress granules appear during virus infection, and their importance is not well understood. Previously, it was assumed that they were nonfunctional artifacts associated with cellular stress. PKR is a well-known antiviral protein; however, its regulation in cells is not well understood. Our work links cellular stress granules with activation of PKR and other innate immune pathways through the activity of G3BP1, a critical stress granule component. The ability of stress granules and G3BP1 to activate PKR and other innate immune transcriptional responses indicates that G3BP1 is an antiviral protein. This work helps to refine a longstanding paradigm indicating stress granules are inert structures and explains why G3BP1 is subverted by many viruses to promote a productive infection.

scholarly journals Small molecules for modulating protein driven liquid-liquid phase separation in treating neurodegenerative disease

2019 ◽  
Author(s):  
Richard J. Wheeler ◽  
Hyun O. Lee ◽  
Ina Poser ◽  
Arun Pal ◽  
Thom Doeleman ◽  
...  
Keyword(s):  
Phase Separation ◽  
Neurodegenerative Disease ◽  
Stress Granules ◽  
Life Time ◽  
Stress Granule ◽  
Phase Behaviour ◽  
Granule Proteins ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractAmyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with few avenues for treatment. Many proteins implicated in ALS associate with stress granules, which are examples of liquid-like compartments formed by phase separation. Aberrant phase transition of stress granules has been implicated in disease, suggesting that modulation of phase transitions could be a possible therapeutic route. Here, we combine cell-based and protein-based screens to show that lipoamide, and its related compound lipoic acid, reduce the propensity of stress granule proteins to aggregate in vitro. More significantly, they also prevented aggregation of proteins over the life time of Caenorhabditis elegans. Observations that they prevent dieback of ALS patient-derived (FUS mutant) motor neuron axons in culture and recover motor defects in Drosophila melanogaster expressing FUS mutants suggest plausibility as effective therapeutics. Our results suggest that altering phase behaviour of stress granule proteins in the cytoplasm could be a novel route to treat ALS.

scholarly journals Retinoic Acid Inducible Gene I and Protein Kinase R, but Not Stress Granules, Mediate the Proinflammatory Response to Yellow Fever Virus

2020 ◽  
Vol 94 (22) ◽  
Author(s):  
Guillaume Beauclair ◽  
Felix Streicher ◽  
Maxime Chazal ◽  
Daniela Bruni ◽  
Sarah Lesage ◽  
...  
Keyword(s):  
Yellow Fever ◽  
Yellow Fever Virus ◽  
Rna Virus ◽  
Stress Granules ◽  
Cytokine Response ◽  
Fever Virus ◽  
Stress Granule ◽  
Proinflammatory Response ◽  
Granule Formation ◽  
Proinflammatory Mediators

ABSTRACT Yellow fever virus (YFV) is an RNA virus primarily targeting the liver. Severe YF cases are responsible for hemorrhagic fever, plausibly precipitated by excessive proinflammatory cytokine response. Pathogen recognition receptors (PRRs), such as the cytoplasmic retinoic acid inducible gene I (RIG-I)-like receptors (RLRs), and the viral RNA sensor protein kinase R (PKR), are known to initiate a proinflammatory response upon recognition of viral genomes. Here, we sought to reveal the main determinants responsible for the acute cytokine expression occurring in human hepatocytes following YFV infection. Using a RIG-I-defective human hepatoma cell line, we found that RIG-I largely contributes to cytokine secretion upon YFV infection. In infected RIG-I-proficient hepatoma cells, RIG-I was localized in stress granules. These granules are large aggregates of stalled translation preinitiation complexes known to concentrate RLRs and PKR and are so far recognized as hubs orchestrating RNA virus sensing. Stable knockdown of PKR in hepatoma cells revealed that PKR contributes to both stress granule formation and cytokine induction upon YFV infection. However, stress granule disruption did not affect the cytokine response to YFV infection, as assessed by small interfering RNA (siRNA)-knockdown-mediated inhibition of stress granule assembly. Finally, no viral RNA was detected in stress granules using a fluorescence in situ hybridization approach coupled with immunofluorescence. Our findings suggest that both RIG-I and PKR mediate proinflammatory cytokine induction in YFV-infected hepatocytes, in a stress granule-independent manner. Therefore, by showing the uncoupling of the cytokine response from the stress granule formation, our model challenges the current view in which stress granules are required for the mounting of the acute antiviral response. IMPORTANCE Yellow fever is a mosquito-borne acute hemorrhagic disease caused by yellow fever virus (YFV). The mechanisms responsible for its pathogenesis remain largely unknown, although increased inflammation has been linked to worsened outcome. YFV targets the liver, where it primarily infects hepatocytes. We found that two RNA-sensing proteins, RIG-I and PKR, participate in the induction of proinflammatory mediators in human hepatocytes infected with YFV. We show that YFV infection promotes the formation of cytoplasmic structures, termed stress granules, in a PKR- but not RIG-I-dependent manner. While stress granules were previously postulated to be essential platforms for immune activation, we found that they are not required for the production of proinflammatory mediators upon YFV infection. Collectively, our work uncovered molecular events triggered by the replication of YFV, which could prove instrumental in clarifying the pathogenesis of the disease, with possible repercussions for disease management.

scholarly journals Stable Formation of Compositionally Unique Stress Granules in Virus-Infected Cells

2009 ◽  
Vol 84 (7) ◽  
pp. 3654-3665 ◽  
Author(s):  
Joanna Piotrowska ◽  
Spencer J. Hansen ◽  
Nogi Park ◽  
Katarzyna Jamka ◽  
Peter Sarnow ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Heat Shock ◽  
Viral Infections ◽  
De Novo ◽  
Stress Granules ◽  
Stress Granule ◽  
Initiation Factor ◽  
Granule Formation ◽  
Infected Cells ◽  
Positive Strand Rna

ABSTRACT Stress granules are sites of mRNA storage formed in response to a variety of stresses, including viral infections. Here, the mechanisms and consequences of stress granule formation during poliovirus infection were examined. The results indicate that stress granules containing T-cell-restricted intracellular antigen 1 (TIA-1) and mRNA are stably constituted in infected cells despite lacking intact RasGAP SH3-domain binding protein 1 (G3BP) and eukaryotic initiation factor 4G. Fluorescent in situ hybridization revealed that stress granules in infected cells do not contain significant amounts of viral positive-strand RNA. Infection does not prevent stress granule formation in response to heat shock, indicating that poliovirus does not block de novo stress granule formation. A mutant TIA-1 protein that prevents stress granule formation during oxidative stress also prevents formation in infected cells. However, stress granule formation during infection is more dependent upon ongoing transcription than is formation during oxidative stress or heat shock. Furthermore, Sam68 is recruited to stress granules in infected cells but not to stress granules formed in response to oxidative stress or heat shock. These results demonstrate that stress granule formation in poliovirus-infected cells utilizes a transcription-dependent pathway that results in the appearance of stable, compositionally unique stress granules.

scholarly journals Cytosolic inactivation of translocated neutrophil plasma membrane protein tyrosine phosphatase

Blood ◽  
1996 ◽  
Vol 87 (1) ◽  
pp. 341-349 ◽  
Author(s):  
Y Cui ◽  
KA Harvey ◽  
RA Siddiqui ◽  
J Jansen ◽  
LP Akard ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Catalytic Activity ◽  
Tyrosine Phosphorylation ◽  
Intracellular Signaling ◽  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Resting Cells ◽  
Gm Csf ◽  
Specific Granules ◽  
Specific Granule

Abstract Phosphotyrosine phosphatases (PTPases) regulate cellular metabolic activation by reversing the effects of tyrosine kinases activated earlier in intracellular signaling pathways. We coupled fluorescence-activated cell sorter analysis using anti-CD45 monoclonal antibody with direct measurements of enzyme activity in resolved subcellular fractions to define mechanisms that potentially regulate the availability and activity of CD45-PTPase on neutrophil plasma membranes. Neutrophils in freshly obtained blood as well as neutrophils freshly isolated from blood were found to possess detectable levels of plasma membrane CD45 as assessed by immunofluorescence. However, plasma membranes from these cells were essentially devoid of PTPase catalytic activity, which was largely confined to the specific granules. Granulocyte-macrophage colony-stimulating factor (GM-CSF) upregulated both the catalytic and antigenic components of CD45-PTPase on the plasma membrane of these cells. Upregulation was associated with a shift in the particulate subcellular PTPase catalytic activity from the specific granule fraction to the plasma membrane fraction. The tyrosine kinase inhibitor genistein abrogated GM-CSF-promoted upregulation of plasma membrane CD45 PTPase but did not prevent the GM-CSF-dependent decrease in specific granule catalytic activity. Anti-CD45 antibody immunoprecipitated PTPase activity from both specific granules of resting cells and plasma membranes of GM-CSF-treated cells. However, antiphosphotyrosine immunoprecipitated only activity that had translocated to the plasma membrane, suggesting a role for CD45 phosphorylation in translocation. Western analysis confirmed the tyrosine phosphorylation of CD45 in plasma membranes of GM-CSF-treated neutrophils. Preincubation of plasma membranes of GM-CSF-stimulated neutrophils with cytosol from resting cells resulted in a time- and temperature-dependent loss in membrane PTPase as a consequence of the effects of a cytosolic inactivator. Cytosol obtained from stimulated neutrophils possessed substantially reduced levels of this PTPase inactivator. We conclude that activity of the catalytic component of membrane PTPase in circulating neutrophils is regulated by a cytosolic inactivator. Upon stimulation, intact CD45 PTPase is incorporated into the plasma membrane by a process that requires tyrosine phosphorylation. As a result of inhibition of the cytosolic inactivator, the translocated PTPase expresses full activity, thereby amplifying the potential regulatory influence of the enzyme on the cells' functional response.

scholarly journals Nuclear stress granules

2004 ◽  
Vol 164 (1) ◽  
pp. 15-17 ◽  
Author(s):  
Anton Sandqvist ◽  
Lea Sistonen
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Heterochromatic Region ◽  
Stress Granules ◽  
Mrna Processing ◽  
Stress Granule ◽  
Target Sites ◽  
Processing Factors

Nuclear stress granules are subnuclear compartments that form in response to heat shock and other stress stimuli. Although many components of nuclear stress granules have been identified, including HSF1 and pre-mRNA processing factors, their function remains a mystery. A paper in this issue describes the stress-induced transcriptional activation of one of the nuclear stress granule target sites, a heterochromatic region that has been considered silent (Jolly et al., 2004). These intriguing findings will certainly give the research of these structures a new twist.

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