scholarly journals Large G3BP-induced granules trigger eIF2α phosphorylation

2012 ◽  
Vol 23 (18) ◽  
pp. 3499-3510 ◽  
Author(s):  
Lucas C. Reineke ◽  
Jon D. Dougherty ◽  
Philippe Pierre ◽  
Richard E. Lloyd
Keyword(s):  
Stress Granules ◽  
Stress Granule ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Translational Machinery ◽  
Initiation Factors ◽  
Eif2α Phosphorylation ◽  
Translation Initiation Factors ◽  
Messenger Ribonucleoprotein ◽  
Translation Apparatus

Stress granules are large messenger ribonucleoprotein (mRNP) aggregates composed of translation initiation factors and mRNAs that appear when the cell encounters various stressors. Current dogma indicates that stress granules function as inert storage depots for translationally silenced mRNPs until the cell signals for renewed translation and stress granule disassembly. We used RasGAP SH3-binding protein (G3BP) overexpression to induce stress granules and study their assembly process and signaling to the translation apparatus. We found that assembly of large G3BP-induced stress granules, but not small granules, precedes phosphorylation of eIF2α. Using mouse embryonic fibroblasts depleted for individual eukaryotic initiation factor 2α (eIF2α) kinases, we identified protein kinase R as the principal kinase that mediates eIF2α phosphorylation by large G3BP-induced granules. These data indicate that increasing stress granule size is associated with a threshold or switch that must be triggered in order for eIF2α phosphorylation and subsequent translational repression to occur. Furthermore, these data suggest that stress granules are active in signaling to the translational machinery and may be important regulators of the innate immune response.

Plant translation initiation factors: it is not easy to be green

2004 ◽  
Vol 32 (4) ◽  
pp. 589-591 ◽  
Author(s):  
K.S. Browning
Keyword(s):  
Translation Initiation ◽  
Binding Proteins ◽  
Initiation Factor ◽  
Eukaryotic Initiation Factor ◽  
Translational Machinery ◽  
Initiation Factors ◽  
Translation Initiation Factors

Plants have significant differences in some of the ‘parts’ of the translational machinery. There are two forms of eukaryotic initiation factor (eIF) 4F, eIF3 has two novel subunits, eIF4B is poorly conserved, and eIF2 kinases and eIF4E binding proteins (4E-BP) are yet to be discovered. These differences suggest that plants may regulate their translation in unique ways.

scholarly journals Uncoupling Stress Granule Assembly and Translation Initiation Inhibition

2009 ◽  
Vol 20 (11) ◽  
pp. 2673-2683 ◽  
Author(s):  
Sophie Mokas ◽  
John R. Mills ◽  
Cristina Garreau ◽  
Marie-Josée Fournier ◽  
Francis Robert ◽  
...  
Keyword(s):  
Translation Initiation ◽  
Binding Protein ◽  
Mrna Translation ◽  
Initiation Factor ◽  
Ribosomal Subunits ◽  
Initiation Factors ◽  
Eif2α Phosphorylation ◽  
Translation Initiation Factors ◽  
Dependent Pathway ◽  
Regulatory Sites

Cytoplasmic stress granules (SGs) are specialized regulatory sites of mRNA translation that form under different stress conditions known to inhibit translation initiation. The formation of SG occurs via two pathways; the eukaryotic initiation factor (eIF) 2α phosphorylation-dependent pathway mediated by stress and the eIF2α phosphorylation-independent pathway mediated by inactivation of the translation initiation factors eIF4A and eIF4G. In this study, we investigated the effects of targeting different translation initiation factors and steps in SG formation in HeLa cells. By depleting eIF2α, we demonstrate that reduced levels of the eIF2.GTP.Met-tRNAiMet ternary translation initiation complexes is sufficient to induce SGs. Likewise, reduced levels of eIF4B, eIF4H, or polyA-binding protein, also trigger SG formation. In contrast, depletion of the cap-binding protein eIF4E or preventing its assembly into eIF4F results in modest SG formation. Intriguingly, interfering with the last step of translation initiation by blocking the recruitment of 60S ribosome either with 2-(4-methyl-2,6-dinitroanilino)-N-methylpropionamideis or through depletion of the large ribosomal subunits protein L28 does not induce SG assembly. Our study identifies translation initiation steps and factors involved in SG formation as well as those that can be targeted without induction of SGs.

scholarly journals Stress Granule Formation Attenuates RACK1-Mediated Apoptotic Cell Death Induced by Morusin

2020 ◽  
Vol 21 (15) ◽  
pp. 5360
Author(s):  
Ye-Jin Park ◽  
Dong Wook Choi ◽  
Sang Woo Cho ◽  
Jaeseok Han ◽  
Siyoung Yang ◽  
...  
Keyword(s):  
Cell Death ◽  
Apoptotic Cell ◽  
Stress Granules ◽  
Protective Role ◽  
Stress Granule ◽  
Protein Kinase R ◽  
Granule Formation ◽  
Protective Function ◽  
Eif2α Phosphorylation

Stress granules are membraneless organelles composed of numerous components including ribonucleoproteins. The stress granules are characterized by a dynamic complex assembly in response to various environmental stressors, which has been implicated in the coordinated regulation of diverse biological pathways, to exert a protective role against stress-induced cell death. Here, we show that stress granule formation is induced by morusin, a novel phytochemical displaying antitumor capacity through barely known mechanisms. Morusin-mediated induction of stress granules requires activation of protein kinase R (PKR) and subsequent eIF2α phosphorylation. Notably, genetic inactivation of stress granule formation mediated by G3BP1 knockout sensitized cancer cells to morusin treatment. This protective function against morusin-mediated cell death can be attributed at least in part to the sequestration of receptors for activated C kinase-1 (RACK1) within the stress granules, which reduces caspase-3 activation. Collectively, our study provides biochemical evidence for the role of stress granules in suppressing the antitumor capacity of morusin, proposing that morusin treatment, together with pharmacological inhibition of stress granules, could be an efficient strategy for targeting cancer.

scholarly journals The Leader Protein of Theiler's Virus Prevents the Activation of PKR

2019 ◽  
Vol 93 (19) ◽  
Author(s):  
Fabian Borghese ◽  
Frédéric Sorgeloos ◽  
Teresa Cesaro ◽  
Thomas Michiels
Keyword(s):  
Stress Granule ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Wild Type Virus ◽  
Granule Formation ◽  
Double Stranded Rna ◽  
Eif2α Phosphorylation ◽  
Leader Protein ◽  
Infected Cells ◽  
L Proteins

ABSTRACT Leader (L) proteins encoded by cardioviruses are multifunctional proteins that contribute to innate immunity evasion. L proteins of Theiler’s murine encephalomyelitis virus (TMEV), Saffold virus (SAFV), and encephalomyocarditis virus (EMCV) were reported to inhibit stress granule assembly in infected cells. Here, we show that TMEV L can act at two levels in the stress granule formation pathway: on the one hand, it can inhibit sodium arsenite-induced stress granule assembly without preventing eIF2α phosphorylation and, thus, acts downstream of eIF2α; on the other hand, it can inhibit eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation and the consequent PKR-mediated eIF2α phosphorylation. Interestingly, coimmunostaining experiments revealed that PKR colocalizes with viral double-stranded RNA (dsRNA) in cells infected with L-mutant viruses but not in cells infected with the wild-type virus. Furthermore, PKR coprecipitated with dsRNA from cells infected with L-mutant viruses significantly more than from cells infected with the wild-type virus. These data strongly suggest that L blocks PKR activation by preventing the interaction between PKR and viral dsRNA. In infected cells, L also rendered PKR refractory to subsequent activation by poly(I·C). However, no interaction was observed between L and either dsRNA or PKR. Taken together, our results suggest that, unlike other viral proteins, L indirectly acts on PKR to negatively regulate its responsiveness to dsRNA. IMPORTANCE The leader (L) protein encoded by cardioviruses is a very short multifunctional protein that contributes to evasion of the host innate immune response. This protein notably prevents the formation of stress granules in infected cells. Using Theiler’s virus as a model, we show that L proteins can act at two levels in the stress response pathway leading to stress granule formation, the most striking one being the inhibition of eucaryotic translation initiation factor 2 alpha kinase 2 (PKR) activation. Interestingly, the leader protein appears to inhibit PKR via a novel mechanism by rendering this kinase unable to detect double-stranded RNA, its typical activator. Unlike other viral proteins, such as influenza virus NS1, the leader protein appears to interact with neither PKR nor double-stranded RNA, suggesting that it acts indirectly to trigger the inhibition of the kinase.

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 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.

Bidirectional RNA helicase activity of eucaryotic translation initiation factors 4A and 4F

1990 ◽  
Vol 10 (3) ◽  
pp. 1134-1144 ◽  
Author(s):  
F Rozen ◽  
I Edery ◽  
K Meerovitch ◽  
T E Dever ◽  
W C Merrick ◽  
...  
Keyword(s):  
Secondary Structure ◽  
Translation Initiation ◽  
Direct Evidence ◽  
Rna Helicase ◽  
Initiation Factor ◽  
Helicase Activity ◽  
Ribosome Binding ◽  
Initiation Factors ◽  
Translation Initiation Factors ◽  
Hydrolysis Of

The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Triggers Apoptosis via Protein Kinase R but Is Resistant to Its Antiviral Activity

2008 ◽  
Vol 83 (5) ◽  
pp. 2298-2309 ◽  
Author(s):  
Verena Krähling ◽  
David A. Stein ◽  
Martin Spiegel ◽  
Friedemann Weber ◽  
Elke Mühlberger
Keyword(s):  
Protein Kinase ◽  
Severe Acute Respiratory Syndrome ◽  
Chromatin Condensation ◽  
Mrna Translation ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Protein Kinase R ◽  
Beta Interferon ◽  
Reverse Transcription Pcr ◽  
Eif2α Phosphorylation

ABSTRACT In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2α (eIF2α). In addition, two of the three cellular eIF2α kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2α phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2α phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2α phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2α on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2α phosphorylation.

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 A Retrospective on eIF2A—and Not the Alpha Subunit of eIF2

2020 ◽  
Vol 21 (6) ◽  
pp. 2054
Author(s):  
Anton A. Komar ◽  
William C. Merrick
Keyword(s):  
Translational Control ◽  
Ribosomal Subunit ◽  
Initiation Factor ◽  
Single Chain ◽  
Initiation Factors ◽  
Eif2α Phosphorylation ◽  
Specific Mrnas ◽  
Internal Initiation ◽  
And Function ◽  
Polypeptide Chains

Initiation of protein synthesis in eukaryotes is a complex process requiring more than 12 different initiation factors, comprising over 30 polypeptide chains. The functions of many of these factors have been established in great detail; however, the precise role of some of them and their mechanism of action is still not well understood. Eukaryotic initiation factor 2A (eIF2A) is a single chain 65 kDa protein that was initially believed to serve as the functional homologue of prokaryotic IF2, since eIF2A and IF2 catalyze biochemically similar reactions, i.e., they stimulate initiator Met-tRNAi binding to the small ribosomal subunit. However, subsequent identification of a heterotrimeric 126 kDa factor, eIF2 (α,β,γ) showed that this factor, and not eIF2A, was primarily responsible for the binding of Met-tRNAi to 40S subunit in eukaryotes. It was found however, that eIF2A can promote recruitment of Met-tRNAi to 40S/mRNA complexes under conditions of inhibition of eIF2 activity (eIF2α-phosphorylation), or its absence. eIF2A does not function in major steps in the initiation process, but is suggested to act at some minor/alternative initiation events such as re-initiation, internal initiation, or non-AUG initiation, important for translational control of specific mRNAs. This review summarizes our current understanding of the eIF2A structure and function.

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