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.     

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.     

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.     

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.     

Translationally repressed mRNA transiently cycles through stress granules during stress

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.     

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.     

Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1DeltaRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.     

Polysome-bound endonuclease PMR1 is targeted to stress granules via stress-specific binding to TIA-1

The generalized process of mRNA decay involves deadenylation followed by release from translating polysomes, decapping, and exonuclease decay of the mRNA body. In contrast the mRNA endonuclease PMR1 forms a selective complex with its translating substrate mRNA, where it initiates decay by cleaving within the mRNA body. In stressed cells the phosphorylation of the alpha subunit of eukaryotic initiation factor 2 causes translating mRNAs to accumulate with stalled 48S subunits in large subcellular structures termed stress granules (SGs), wherein mRNAs undergo sorting for reinitiation, storage, or decay. Given the unique relationship between translation and PMR1-mediated mRNA decay, we examined the impact of stress-induced dissociation of polysomes on this process. Arsenite stress disrupts the polysome binding of PMR1 and its substrate mRNA but has no impact on the critical tyrosine phosphorylation of PMR1, its association with substrate mRNA, or its association with the functional approximately 680-kDa mRNP complex in which it normally resides on polysomes. We show that arsenite stress drives PMR1 into an RNase-resistant complex with TIA-1, and we identify a distinct domain in the N terminus of PMR1 that facilitates its interaction with TIA-1. Finally, we show that arsenite promotes the delayed association of PMR1 with SGs under conditions which cause tristetraprolin and butyrate response factor 1, proteins that facilitate exonucleolytic mRNA, to exit SGs.     

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.     

Relief of microRNA-mediated translational repression in human cells subjected to stress

In metazoans, most microRNAs imperfectly base-pair with the 3' untranslated region (3'UTR) of target mRNAs and prevent protein accumulation by either repressing translation or inducing mRNA degradation. Examples of specific mRNAs undergoing microRNA-mediated repression are numerous, but whether the repression is a reversible process remains largely unknown. Here we show that cationic amino acid transporter 1 (CAT-1) mRNA and reporters bearing its 3'UTR can be relieved from the microRNA miR-122-induced inhibition in human hepatocarcinoma cells subjected to different stress conditions. The derepression of CAT-1 mRNA is accompanied by its release from cytoplasmic processing bodies and its recruitment to polysomes. The derepression requires binding of HuR, an AU-rich-element binding protein, to the 3'UTR of CAT-1 mRNA. We propose that proteins interacting with the 3'UTR will generally act as modifiers altering the potential of miRNAs to repress gene expression.     

Interactions between brome mosaic virus RNAs and cytoplasmic processing bodies

Cytoplasmic processing bodies are sites where nontranslating mRNAs accumulate for different fates, including decapping and degradation, storage, or returning to translation. Previous work has also shown that the Lsm1-7p complex, Dhh1p, and Pat1p, which are all components of P bodies, are required for translation and subsequent recruitment to replication of the plant virus brome mosaic virus (BMV) genomic RNAs when replication is reproduced in yeast cells. To better understand the role of P bodies in BMV replication, we examined the subcellular locations of BMV RNAs in yeast cells. We observed that BMV genomic RNA2 and RNA3 accumulated in P bodies in a manner dependent on cis-acting RNA replication signals, which also directed nonviral RNAs to P bodies. Furthermore, the viral RNA-dependent RNA polymerase coimmunoprecipitates and shows partial colocalization with the P-body component Lsm1p. These observations suggest that the accumulation of BMV RNAs in P bodies may be an important step in RNA replication complex assembly for BMV, and possibly for other positive-strand RNA viruses.