Dcp2 phosphorylation by Ste20 modulates stress granule assembly and mRNA decay in Saccharomyces cerevisiae

Translation and messenger RNA (mRNA) degradation are important sites of gene regulation, particularly during stress where translation and mRNA degradation are reprogrammed to stabilize bulk mRNAs and to preferentially translate mRNAs required for the stress response. During stress, untranslating mRNAs accumulate both in processing bodies (P-bodies), which contain some translation repressors and the mRNA degradation machinery, and in stress granules, which contain mRNAs stalled in translation initiation. How signal transduction pathways impinge on proteins modulating P-body and stress granule formation and function is unknown. We show that during stress in Saccharomyces cerevisiae, Dcp2 is phosphorylated on serine 137 by the Ste20 kinase. Phosphorylation of Dcp2 affects the decay of some mRNAs and is required for Dcp2 accumulation in P-bodies and specific protein interactions of Dcp2 and for efficient formation of stress granules. These results demonstrate that Ste20 has an unexpected role in the modulation of mRNA decay and translation and that phosphorylation of Dcp2 is an important control point for mRNA decapping.     

GW body disassembly triggered by siRNAs independently of their silencing activity

GW bodies (or P-bodies) are cytoplasmic granules containing proteins involved in both mRNA degradation and storage, including the RNA interference machinery. Their mechanism of assembly and function are still poorly known although their number depends upon the flux of mRNA to be stored or degraded. We show here that silencing of the translational regulator CPEB1 leads to their disappearance, as reported for other GW body components. Surprisingly, the same results were obtained with several siRNAs targeting genes encoding proteins unrelated to mRNA metabolism. The disappearance of GW bodies did not correlate with the silencing activity of the siRNA and did not inhibit further silencing by siRNA. Importantly, in most cases, GW bodies were rapidly reinduced by arsenite, indicating that their assembly was not prevented by the inhibition of the targeted or off-target genes. We therefore propose that some siRNA sequences affect mRNA metabolism so as to diminish the amount of mRNA directed to the GW bodies. As an exception, GW bodies were not reinduced following Rck/p54 depletion by interference, indicating that this component is truly required for the GW body assembly. Noteworthy, Rck/p54 was dispensable for the assembly of stress granules, in spite of their close relationship with the GW bodies.     

P bodies, stress granules, and viral life cycles

Eukaryotic mRNAs are in a dynamic equilibrium between different subcellular locations. Translating mRNAs can be found in polysomes, mRNAs stalled in translation initiation accumulate in stress granules and mRNAs targeted for degradation or translation repression can accumulate in P bodies. Partitioning of mRNAs between polysomes, stress granules, and P bodies affects rates of translation and mRNA degradation. Host proteins within P bodies and stress granules can enhance or limit viral infection, and some viral RNAs and proteins accumulate in P bodies and/or stress granules. Thus, an important interplay among P bodies, stress granules, and viral life cycles is beginning to emerge.     

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.     

Polyamines inhibit the assembly of stress granules in normal intestinal epithelial cells regulating apoptosis

Polyamines regulate multiple signaling pathways and are implicated in many aspects of cellular functions, but the exact molecular processes governed by polyamines remain largely unknown. In response to environmental stress, repression of translation is associated with the assembly of stress granules (SGs) that contain a fraction of arrested mRNAs and are thought to function as mRNA storage. Here we show that polyamines modulate the assembly of SGs in normal intestinal epithelial cells (IECs) and that induced SGs following polyamine depletion are implicated in the protection of IECs against apoptosis. Increasing the levels of cellular polyamines by ectopic overexpression of the ornithine decarboxylase gene decreased cytoplasmic levels of SG-signature constituent proteins eukaryotic initiation factor 3b and T-cell intracellular antigen-1 (TIA-1)-related protein and repressed the assembly of SGs induced by exposure to arsenite-induced oxidative stress. In contrast, depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine increased cytoplasmic eukaryotic initiation factor 3b and TIA-1 related protein abundance and enhanced arsenite-induced SG assembly. Polyamine-deficient cells also exhibited an increase in resistance to tumor necrosis factor-α/cycloheximide-induced apoptosis, which was prevented by inhibiting SG formation with silencing SG resident proteins Sort1 and TIA-1. These results indicate that the elevation of cellular polyamines represses the assembly of SGs in normal IECs and that increased SGs in polyamine-deficient cells are crucial for increased resistance to apoptosis.     

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.     

P bodies promote stress granule assembly in Saccharomyces cerevisiae

Recent results indicate that nontranslating mRNAs in eukaryotic cells exist in distinct biochemical states that accumulate in P bodies and stress granules, although the nature of interactions between these particles is unknown. We demonstrate in Saccharomyces cerevisiae that RNA granules with similar protein composition and assembly mechanisms as mammalian stress granules form during glucose deprivation. Stress granule assembly is dependent on P-body formation, whereas P-body assembly is independent of stress granule formation. This suggests that stress granules primarily form from mRNPs in preexisting P bodies, which is also supported by the kinetics of P-body and stress granule formation both in yeast and mammalian cells. These observations argue that P bodies are important sites for decisions of mRNA fate and that stress granules, at least in yeast, primarily represent pools of mRNAs stalled in the process of reentry into translation from P bodies.     

Poliovirus-mediated disruption of cytoplasmic processing bodies

Metazoan cells form cytoplasmic mRNA granules such as stress granules (SG) and processing bodies (P bodies) that are proposed to be sites of aggregated, translationally silenced mRNAs and mRNA degradation. Poliovirus (PV) is a plus-strand RNA virus containing a genome that is a functional mRNA; thus, we investigated if PV antagonizes the processes that lead to formation of these structures. We have previously shown that PV infection inhibits the ability of cells to form stress granules by cleaving RasGAP-SH3-binding protein (G3BP). Here, we show that P bodies are also disrupted during PV infection in cells by 4 h postinfection. The disruption of P bodies is more rapid and more complete than disruption of stress granules. The kinetics of P body disruption correlated with production of viral proteinases and required substantial viral gene product expression. The organizing mechanism that forms P body foci in cells is unknown; however, potential scaffolding, aggregating, or other regulatory proteins found in P bodies were investigated for degradation. Two factors involved in 5'-end mRNA decapping and degradation, Xrn1 and Dcp1a, and the 3' deadenylase complex component Pan3 underwent accelerated degradation during infection, and Dcp1a may be a direct substrate of PV 3C proteinase. Several other key factors proposed to be essential for P body formation, GW182, Edc3, and Edc4, were unaffected by poliovirus infection. Since deadenylation has been reported to be required for P body formation, viral inhibition of deadenylation, through Pan3 degradation, is a potential mechanism of P body disruption.     

Myosin Va is required for P body but not stress granule formation

In the present study we demonstrate an association between mammalian myosin Va and cytoplasmic P bodies, microscopic ribonucleoprotein granules that contain components of the 5'-3' mRNA degradation machinery. Myosin Va colocalizes with several P body markers and its RNAi-mediated knockdown results in the disassembly of P bodies. Overexpression of a dominant-negative mutant of myosin Va reduced the motility of P bodies in living cells. Co-immunoprecipitation experiments demonstrate that myosin Va physically associates with eIF4E, an mRNA binding protein that localizes to P bodies. In contrast, we find that myosin Va does not play a role in stress granule formation. Stress granules are ribonucleoprotein structures that are involved in translational silencing and are spatially, functionally, and compositionally linked to P bodies. Myosin Va is found adjacent to stress granules in stressed cells but displays minimal localization within stress granules, and myosin Va knockdown has no effect on stress granule assembly or disassembly. Combined with recently published reports demonstrating a role for Drosophila and mammalian class V myosins in mRNA transport and the involvement of the yeast myosin V orthologue Myo2p in P body assembly, our results provide further evidence that the class V myosins serve an important role in the transport and turnover of mRNA.     

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.     

ZBP1 regulates mRNA stability during cellular stress

An essential constituent of the integrated stress response (ISR) is a reversible translational suppression. This mRNA silencing occurs in distinct cytoplasmic foci called stress granules (SGs), which transiently associate with processing bodies (PBs), typically serving as mRNA decay centers. How mRNAs are protected from degradation in these structures remains elusive. We identify that Zipcode-binding protein 1 (ZBP1) regulates the cytoplasmic fate of specific mRNAs in nonstressed cells and is a key regulator of mRNA turnover during the ISR. ZBP1 association with target mRNAs in SGs was not essential for mRNA targeting to SGs. However, ZBP1 knockdown induced a selective destabilization of target mRNAs during the ISR, whereas forced expression increased mRNA stability. Our results indicate that although targeting of mRNAs to SGs is nonspecific, the stabilization of mRNAs during cellular stress requires specific protein-mRNA interactions. These retain mRNAs in SGs and prevent premature decay in PBs. Hence, mRNA-binding proteins are essential for translational adaptation during cellular stress by modulating mRNA turnover.     

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.     

Large P body-like RNPs form in C. elegans oocytes in response to arrested ovulation, heat shock, osmotic stress, and anoxia and are regulated by the major sperm protein pathway

As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.     

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.     

Role of Microtubules in Stress Granule Assembly: MICROTUBULE DYNAMICAL INSTABILITY FAVORS THE FORMATION OF MICROMETRIC STRESS GRANULES IN CELLS*

Following exposure to various stresses (arsenite, UV, hyperthermia, and hypoxia), mRNAs are assembled into large cytoplasmic bodies known as “stress granules,” in which mRNAs and associated proteins may be processed by specific enzymes for different purposes like transient storing, sorting, silencing, or other still unknown processes. To limit mRNA damage during stress, the assembly of micrometric granules has to be rapid, and, indeed, it takes only ∼10–20 min in living cells. However, such a rapid assembly breaks the rules of hindered diffusion in the cytoplasm, which states that large cytoplasmic bodies are almost immobile. In the present work, using HeLa cells and YB-1 protein as a stress granule marker, we studied three hypotheses to understand how cells overcome the limitation of hindered diffusion: shuttling of small messenger ribonucleoprotein particles from small to large stress granules, sliding of messenger ribonucleoprotein particles along microtubules, microtubule-mediated stirring of large stress granules. Our data favor the two last hypotheses and underline that microtubule dynamic instability favors the formation of micrometric stress granules.     

Dcp1-Bodies in Mouse Oocytes

Processing bodies (P-bodies) are cytoplasmic granules involved in the storage and degradation of mRNAs. In somatic cells, their formation involves miRNA-mediated mRNA silencing. Many P-body protein components are also found in germ cell granules, such as in mammalian spermatocytes. In fully grown mammalian oocytes, where changes in gene expression depend entirely on translational control, RNA granules have not as yet been characterized. Here we show the presence of P-body-like foci in mouse oocytes, as revealed by the presence of Dcp1a and the colocalization of RNA-associated protein 55 (RAP55) and the DEAD box RNA helicase Rck/p54, two proteins associated with P-bodies and translational control. These P-body-like structures have been called Dcp1-bodies and in meiotically arrested primary oocytes, two types can be distinguished based on their size. They also have different protein partners and sensitivities to the depletion of endogenous siRNA/miRNA and translational inhibitors. However, both type progressively disappear during in vitro meiotic maturation and are virtually absent in metaphase II–arrested secondary oocytes. Moreover, this disassembly of hDcp1a-bodies is concomitant with the posttranslational modification of EGFP-hDcp1a.