Inhibition of the ubiquitin-proteasome system induces stress granule formation

The inhibition of the ubiquitin-dependent proteasome system (UPS) via specific drugs is one type of approach used to combat cancer. Although it has been suggested that UPS inhibition prevents the rapid decay of AU-rich element (ARE)-containing messages, very little is known about the cellular mechanisms leading to this effect. Here we establish a link between the inhibition of UPS activity, the formation of cytoplasmic stress granules (SGs), and mRNA metabolism. The assembly of the SGs requires the phosphorylation of the translation initiation factor eIF2alpha by a mechanism involving the stress kinase GCN2. On prolonged UPS inhibition and despite the maintenance of eIF2alpha phosphorylation, SGs disassemble and translation recovers in an Hsp72 protein-dependent manner. The formation of these SGs coincides with the disassembly of processing bodies (PBs), known as mRNA decay entities. As soon as the SGs assemble, they recruit ARE-containing messages such as p21(cip1) mRNA, which are stabilized under these conditions. Hence, our findings suggest that SGs could be considered as one of the players that mediate the early response of the cell to proteasome inhibitors by interfering temporarily with mRNA decay pathways.     

Novel mRNA-containing cytoplasmic granules in ALK-transformed cells

In mammalian cells, nontranslating messenger RNAs (mRNAs) are concentrated in different cytoplasmic foci, such as processing bodies (PBs) and stress granules (SGs), where they are either degraded or stored. In the present study, we have thoroughly characterized cytoplasmic foci, hereafter called AGs for ALK granules that form in transformed cells expressing the constitutively active anaplastic lymphoma kinase (ALK). AGs contain polyadenylated mRNAs and a unique combination of several RNA binding proteins that so far has not been described in mammalian foci, including AUF1, HuR, and the poly (A(+)) binding protein PABP. AGs shelter neither components of the mRNA degradation machinery present in PBs nor known markers of SGs, such as translation initiation factors or TIA/TIAR, showing that they are distinct from PBs or SGs. AGs and PBs, however, both move on microtubules with similar dynamics and frequently establish close contacts. In addition, in conditions in which mRNA metabolism is perturbed, AGs concentrate PB components with the noticeable exception of the 5' to 3' exonuclease XRN1. Altogether, we show that AGs constitute novel mRNA-containing cytoplasmic foci and we propose that they could protect translatable mRNAs from degradation, contributing thus to ALK-mediated oncogenicity.     

Tyrosine phosphorylation of HuR by JAK3 triggers dissociation and degradation of HuR target mRNAs

In response to stress conditions, many mammalian mRNAs accumulate in stress granules (SGs) together with numerous RNA-binding proteins that control mRNA turnover and translation. However, the signaling cascades that modulate the presence of ribonucleoprotein (RNP) complexes in SGs are poorly understood. Here, we investigated the localization of human antigen R (HuR), an mRNA-stabilizing RNA-binding protein, in SGs following exposure to the stress agent arsenite. Unexpectedly, the mobilization of HuR to SGs was prevented through the activation of Janus kinase 3 (JAK3) by the vitamin K3 analog menadione. JAK3 phosphorylated HuR at tyrosine 200, in turn inhibiting HuR localization in SGs, reducing HuR interaction with targets SIRT1 and VHL mRNAs, and accelerating target mRNA decay. Our findings indicate that HuR is tyrosine-phosphorylated by JAK3, and link this modification to HuR subcytoplasmic localization and to the fate of HuR target mRNAs.     

Yeast processing bodies and stress granules: self-assembly ribonucleoprotein particles

Processing bodies (PBs) and stress granules (SGs) are two highly conserved cytoplasmic ribonucleoprotein foci that contain translationally repressed mRNAs together with proteins from the mRNA metabolism. Interestingly, they also share some common features with other granules, including the prokaryotic inclusion bodies. Although the function of PBs and SGs remains elusive, major advances have been done in unraveling their composition and assembly by using the yeast Saccharomyces cerevisae.     

Phosphorylation of the Eukaryotic Translation Initiation Factor 4E-Transporter (4E-T) by c-Jun N-Terminal Kinase Promotes Stress-Dependent P-Body Assembly

Processing bodies (PBs, or P bodies) are cytoplasmic granules involved in mRNA storage and degradation that participate in the regulation of gene expression. PBs concentrate nontranslated mRNAs and several factors involved in mRNA decay and translational repression, including the eukaryotic translation initiation factor 4E-transporter (4E-T). 4E-T is required for PB assembly, but little is known about the molecular mechanisms that regulate its function. Here, we demonstrate that oxidative stress promotes multisite 4E-T phosphorylation. We show that the c-Jun N-terminal kinase (JNK) is targeted to PBs in response to oxidative stress and promotes the phosphorylation of 4E-T. Quantitative mass spectrometry analysis reveals that JNK phosphorylates 4E-T on six proline-directed sites that are required for the formation of the 4E-T complex upon stress. We have developed an image-based computational method to quantify the size, number, and density of PBs in cells, and we find that while 4E-T is required for steady-state PB assembly, its phosphorylation facilitates the formation of larger PBs upon oxidative stress. Using polysomal mRNA profiling, we assessed global and specific mRNA translation but did not find that 4E-T phosphorylation impacts translational control. Collectively, these data support a model whereby PB assembly is regulated by a two-step mechanism involving a 4E-T-dependent assembly stage in unstressed cells and a 4E-T phosphorylation-dependent aggregation stage in response to stress stimuli.     

Metazoan stress granule assembly is mediated by P-eIF2α-dependent and -independent mechanisms

Stress granules (SGs) are cytoplasmic bodies wherein translationally silenced mRNAs are recruited for triage in response to environmental stress. We report that Drosophila cells form SGs in response to arsenite and heat shock. Drosophila SGs, like mammalian SGs, are distinct from but adjacent to processing bodies (PBs, sites of mRNA silencing and decay), require polysome disassembly, and are in dynamic equilibrium with polysomes. We further examine the role of the two Drosophila eIF2α kinases, PEK and GCN2, in regulating SG formation in response to heat and arsenite stress. While arsenite-induced SGs are dependent upon eIF2α phosphorylation, primarily via PEK, heat-induced SGs are phospho-eIF2α-independent. In contrast, heat-induced SGs require eIF2α phosphorylation in mammalian cells, as non-phosphorylatable eIF2α Ser51Ala mutant murine embryonic fibroblasts do not form SGs even after severe heat shock. These results suggest that mammals evolved alternative mechanisms for dealing with thermal stress.     

Stress granules are dispensable for mRNA stabilization during cellular stress

During cellular stress, protein synthesis is severely reduced and bulk mRNA is recruited to stress granules (SGs). Previously, we showed that the SG-recruited IGF2 mRNA-binding protein 1 (IGF2BP1) interferes with target mRNA degradation during cellular stress. Whether this requires the formation of SGs remained elusive. Here, we demonstrate that the sustained inhibition of visible SGs requires the concomitant knockdown of TIA1, TIAR and G3BP1. FRAP and photo-conversion studies, however, indicate that these proteins only transiently associate with SGs. This suggests that instead of forming a rigid scaffold for mRNP recruitment, TIA proteins and G3BP1 promote SG-formation by constantly replenishing mRNPs. In contrast, RNA-binding proteins like IGF2BP1 or HUR, which are dispensable for SG-assembly, are stably associated with SGs and the IGF2BP1/HUR-G3BP1 association is increased during stress. The depletion of IGF2BP1 enhances the degradation of target mRNAs irrespective of inhibiting SG-formation, whereas the turnover of bulk mRNA remains unaffected when SG-formation is impaired. Together these findings indicate that the stabilization of mRNAs during cellular stress is facilitated by the formation of stable mRNPs, which are recruited to SGs by TIA proteins and/or G3BP1. Importantly, however, the aggregation of mRNPs to visible SGs is dispensable for preventing mRNA degradation.     

Cytoplasmic hGle1A regulates stress granules by modulation of translation

When eukaryotic cells respond to stress, gene expression pathways change to selectively export and translate subsets of mRNAs. Translationally repressed mRNAs accumulate in cytoplasmic foci known as stress granules (SGs). SGs are in dynamic equilibrium with the translational machinery, but mechanisms controlling this are unclear. Gle1 is required for DEAD-box protein function during mRNA export and translation. We document that human Gle1 (hGle1) is a critical regulator of translation during stress. hGle1 is recruited to SGs, and hGLE1 small interfering RNA–mediated knockdown perturbs SG assembly, resulting in increased numbers of smaller SGs. The rate of SG disassembly is also delayed. Furthermore, SG hGle1-depletion defects correlate with translation perturbations, and the hGle1 role in SGs is independent of mRNA export. Interestingly, we observe isoform-specific roles for hGle1 in which SG function requires hGle1A, whereas mRNA export requires hGle1B. We find that the SG defects in hGle1-depleted cells are rescued by puromycin or DDX3 expression. Together with recent links of hGLE1 mutations in amyotrophic lateral sclerosis patients, these results uncover a paradigm for hGle1A modulating the balance between translation and SGs during stress and disease.     

Characterizing mRNA interactions with RNA granules during translation initiation inhibition

When cells experience environmental stresses, global translational arrest is often accompanied by the formation of stress granules (SG) and an increase in the number of p-bodies (PBs), which are thought to play a crucial role in the regulation of eukaryotic gene expression through the control of mRNA translation and degradation. SGs and PBs have been extensively studied from the perspective of their protein content and dynamics but, to date, there have not been systematic studies on how they interact with native mRNA granules. Here, we demonstrate the use of live-cell hybridization assays with multiply-labeled tetravalent RNA imaging probes (MTRIPs) combined with immunofluorescence, as a tool to characterize the polyA+ and β-actin mRNA distributions within the cytoplasm of epithelial cell lines, and the changes in their colocalization with native RNA granules including SGs, PBs and the RNA exosome during the inhibition of translational initiation. Translation initiation inhibition was achieved via the induction of oxidative stress using sodium arsenite, as well as through the use of Pateamine A, puromycin and cycloheximide. This methodology represents a valuable tool for future studies of mRNA trafficking and regulation within living cells.     

Plant stress granules and mRNA processing bodies are distinct from heat stress granules

Similar to the situation in mammalian cells and yeast, messenger ribonucleo protein (mRNP) homeostasis in plant cells depends on rapid transitions between three functional states, i.e. translated mRNPs in polysomes, stored mRNPs and mRNPs under degradation. Studies in mammalian cells showed that whenever the dynamic exchange of the components between these states is disrupted, stalled mRNPs accumulate in cytoplasmic aggregates, such as stress granules (SGs) or processing bodies (PBs). We identified PBs and SGs in plant cells by detection of DCP1, DCP2 and XRN4, as marker proteins for the 5'-->3' mRNA degradation pathway, and eIF4E, as well as the RNA binding proteins RBP47 and UBP1, as marker proteins for stored mRNPs in SGs. Cycloheximide-inhibited translation, stress treatments and mutants defective in mRNP homeostasis were used to study the dynamic transitions of mRNPs between SGs and PBs. SGs and PBs can be clearly discriminated from the previously described heat stress granules (HSGs), which evidently do not contain mRNPs. Thus, the role of HSGs as putative mRNP storage sites must be revised.     

Intracellular trafficking and dynamics of P bodies

RNAs are exported from the nucleus to the cytoplasm, where they undergo translation and produce proteins needed for the cellular life cycle. Some mRNAs are targeted by different RNA decay mechanisms and thereby undergo degradation. The 5’→3’ degradation machinery localizes to cytoplasmic complexes termed P bodies (PBs). They function in RNA turnover, translational repression, RNA-mediated silencing, and RNA storage. A quantitative live-cell imaging approach to study the dynamic aspects of PB trafficking in the cytoplasm revealed that PB movements are rather confined and dependent on an existing microtubule network. Microtubule depolymerization led to a drastic decrease in PB mobility, as well as a release of regulation on PB assembly and a dramatic increase in PB numbers. The different aspects of PB trafficking and encounters with mRNA molecules in the cytoplasm are discussed.     

Recruitment of the RNA helicase RHAU to stress granules via a unique RNA-binding domain

In response to environmental stress, the translation machinery of cells is reprogrammed. The majority of actively translated mRNAs are released from polysomes and driven to specific cytoplasmic foci called stress granules (SGs) where dynamic changes in protein-RNA interaction determine the subsequent fate of mRNAs. Here we show that the DEAH box RNA helicase RHAU is a novel SG-associated protein. Although RHAU protein was originally identified as an AU-rich element-associated protein involved in urokinase-type plasminogen activator mRNA decay, it was not clear whether RHAU could directly interact with RNA. We have demonstrated that RHAU physically interacts with RNA in vitro and in vivo through a newly identified N-terminal RNA-binding domain, which was found to be both essential and sufficient for RHAU localization in SGs. We have also shown that the ATPase activity of RHAU plays a role in the RNA interaction and in the regulation of protein retention in SGs. Thus, our results show that RHAU is the fourth RNA helicase detected in SGs, after rck/p54, DDX3, and eIF4A, and that its association with SGs is dynamic and mediated by an RHAU-specific RNA-binding domain.     

Human hnRNP Q re-localizes to cytoplasmic granules upon PMA, thapsigargin, arsenite and heat-shock treatments

Eukaryotic gene expression is regulated on different levels ranging from pre-mRNA processing to translation. One of the most characterized families of RNA-binding proteins is the group of hnRNPs: heterogenous nuclear ribonucleoproteins. Members of this protein family play important roles in gene expression control and mRNAs metabolism. In the cytoplasm, several hnRNPs proteins are involved in RNA-related processes and they can be frequently found in two specialized structures, known as GW-bodies (GWbs), previously known as processing bodies: PBs, and stress granules, which may be formed in response to specific stimuli. GWbs have been early reported to be involved in the mRNA decay process, acting as a site of mRNA degradation. In a similar way, stress granules (SGs) have been described as cytoplasmic aggregates, which contain accumulated mRNAs in cells under stress conditions and present reduced or inhibited translation. Here, we characterized the hnRNP Q localization after different stress conditions. hnRNP Q is a predominantly nuclear protein that exhibits a modular organization and several RNA-related functions. Our data suggest that the nuclear localization of hnRNP Q might be modified after different treatments, such as: PMA, thapsigargin, arsenite and heat shock. Under different stress conditions, hnRNP Q can fully co-localize with the endoplasmatic reticulum specific chaperone, BiP. However, under stress, this protein only co-localizes partially with the proteins: GW182 — GWbs marker protein and TIA-1 stress granule component.     

RNA-binding proteins TIA-1 and TIAR link the phosphorylation of eIF-2 alpha to the assembly of mammalian stress granules

In response to environmental stress, the related RNA-binding proteins TIA-1 and TIAR colocalize with poly(A)(+) RNA at cytoplasmic foci that resemble the stress granules (SGs) that harbor untranslated mRNAs in heat shocked plant cells (Nover et al. 1989; Nover et al. 1983; Scharf et al. 1998). The accumulation of untranslated mRNA at SGs is reversible in cells that recover from a sublethal stress, but irreversible in cells subjected to a lethal stress. We have found that the assembly of TIA-1/R(+) SGs is initiated by the phosphorylation of eIF-2alpha. A phosphomimetic eIF-2alpha mutant (S51D) induces the assembly of SGs, whereas a nonphosphorylatable eIF-2alpha mutant (S51A) prevents the assembly of SGs. The ability of a TIA-1 mutant lacking its RNA-binding domains to function as a transdominant inhibitor of SG formation suggests that this RNA-binding protein acts downstream of the phosphorylation of eIF-2alpha to promote the sequestration of untranslated mRNAs at SGs. The assembly and disassembly of SGs could regulate the duration of stress- induced translational arrest in cells recovering from environmental stress.     

The Cth2 ARE-binding protein recruits the Dhh1 helicase to promote the decay of succinate dehydrogenase SDH4 mRNA in response to iron deficiency

Iron is an essential nutrient that participates as a redox co-factor in a broad range of cellular processes. In response to iron deficiency, the budding yeast Saccharomyces cerevisiae induces the expression of the Cth1 and Cth2 mRNA-binding proteins to promote a genome-wide remodeling of cellular metabolism that contributes to the optimal utilization of iron. Cth1 and Cth2 proteins bind to specific AU-rich elements within the 3'-untranslated region of many mRNAs encoding proteins involved in iron-dependent pathways, thereby promoting their degradation. Here, we show that the DEAD box Dhh1 helicase plays a crucial role in the mechanism of Cth2-mediated mRNA turnover. Yeast two-hybrid experiments indicate that Cth2 protein interacts in vivo with the carboxyl-terminal domain of Dhh1. We demonstrate that the degradation of succinate dehydrogenase SDH4 mRNA, a known target of Cth2 on iron-deficient conditions, depends on Dhh1. In addition, we localize the Cth2 protein to cytoplasmic processing bodies in strains defective in the 5' to 3' mRNA decay pathway. Finally, the degradation of trapped SDH4 mRNA intermediates by Cth2 supports the 5' to 3' directionality of mRNA turnover. Taken together, these results suggest that Cth2 protein recruits the Dhh1 helicase to ARE-containing mRNAs to promote mRNA decay.