Processing bodies and germ granules are distinct RNA granules that interact in C. elegans embryos

In somatic cells, untranslated mRNAs accumulate in cytoplasmic foci called processing bodies or P-bodies. P-bodies contain complexes that inhibit translation and stimulate mRNA deadenylation, decapping, and decay. Recently, certain P-body proteins have been found in germ granules, RNA granules specific to germ cells. We have investigated a possible connection between P-bodies and germ granules in Caenorhabditis elegans. We identify PATR-1, the C. elegans homolog of the yeast decapping activator Pat1p, as a unique marker for P-bodies in C. elegans embryos. We find that P-bodies are inherited maternally as core granules that mature differently in somatic and germline blastomeres. In somatic blastomeres, P-bodies recruit the decapping activators LSM-1 and LSM-3. This recruitment requires the LET-711/Not1 subunit of the CCR4-NOT deadenylase and correlates spatially and temporally with the onset of maternal mRNA degradation. In germline blastomeres, P-bodies are maintained as core granules lacking LSM-1 and LSM-3. P-bodies interact with germ granules, but maintain distinct dynamics and components. The maternal mRNA nos-2 is maintained in germ granules, but not in P-bodies. We conclude that P-bodies are distinct from germ granules, and represent a second class of RNA granules that behaves differently in somatic and germline cells.     

The nuclear pore complex and the DEAD box protein Rat8p/Dbp5p have nonessential features which appear to facilitate mRNA export following heat shock

Nuclear pore complexes (NPCs) play an essential role in RNA export. Nucleoporins required for mRNA export in Saccharomyces cerevisiae are found in the Nup84p and Nup82p subcomplexes of the NPC. The Nup82p subcomplex contains Nup82p, Rat7p/Nup159p, Nsp1p, Gle1p/Rss1p, and Rip1p/Nup42p and is found only on the cytoplasmic face of NPCs. Both Rat7p and Gle1p contain binding sites for Rat8p/Dbp5p, an essential DEAD box protein and putative RNA helicase. Rip1p interacts directly with Gle1p and is the only protein known to be essential for mRNA export after heat shock but not under normal growth conditions. We report that in cells lacking Rip1p, both Gle1p and Rat8p dissociate from NPCs following heat shock at 42 degrees C. Rat8p but not Gle1p was retained at NPCs if rip1Delta cells were first shifted to 37 degrees C and then to 42 degrees C, and this was correlated with preserving mRNA export in heat-shocked rip1Delta cells. Export following ethanol shock was less dependent on the presence of Rip1p. Exposure to 10% ethanol led to dissociation of Rat8p from NPCs in both wild-type and rip1Delta cells. Following this treatment, Rat8p was primarily nuclear in wild-type cells but primarily cytoplasmic in rip1Delta cells. We also determined that efficient export of heat shock mRNA after heat shock depends upon a novel 6-amino-acid element within Rat8p. This motif is not required under normal growth conditions or following ethanol shock. These studies suggest that the molecular mechanism responsible for the defect in export of heat shock mRNAs in heat-shocked rip1Delta cells is dissociation of Rat8p from NPCs. These studies also suggest that both nuclear pores and Rat8p have features not required for mRNA export in growing cells but which enhance the ability of mRNAs to be exported following heat shock.     

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.     

Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export.

To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions. We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking). Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation. Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p. Temperature-sensitive alleles of DBP5/RAT8 were prepared. In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature. Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region. Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs). The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.     

A conditional allele of the novel repeat-containing yeast nucleoporin RAT7/NUP159 causes both rapid cessation of mRNA export and reversible clustering of nuclear pore complexes

In a screen for Saccharomyces cerevisiae genes required for nucleocytoplasmic transport of messenger RNA, we identified the RAT7 gene (ribonucleic acid trafficking), which encodes an essential protein of 1,460 amino acids. Rat7p is located at the nuclear rim in a punctate pattern characteristic of nucleoporins. Furthermore, the central third of Rat7p contains 22 XXFG and three XFXFG degenerate repeats that are similar to signature GLFG and XFXFG repeats present in a majority of yeast and some mammalian nucleoporins sequenced to date. Shift of a strain bearing the temperature-sensitive rat7-1 allele from 23 degrees C to 37 degrees C resulted in rapid (within 15 minutes) cessation of mRNA export, but did not cause concomitant cytoplasmic accumulation of a reporter protein bearing a nuclear localization signal. This suggests that Rat7p may play a direct role in nucleocytoplasmic export of RNA. Immunofluorescence and thin section electron microscopy revealed that in rat7-1 cells grown at 23 degrees C, the majority of nuclear pore complexes (NPCs) were clustered on one side of the nucleus. No ultrastructural abnormalities of the nuclear envelope were seen. Interestingly, shifting rat7-1 cells to 37 degrees C for 1 h caused the NPCs to disperse, restoring near wild-type NPC distribution. After this temperature shift, the mutant Rat7p was no longer detectable by immunofluorescence.     

The product of the Saccharomyces cerevisiae RSS1 gene, identified as a high-copy suppressor of the rat7-1 temperature-sensitive allele of the RAT7/NUP159 nucleoporin, is required for efficient mRNA export.

RAT7/NUP159 was identified previously in a screen for genes whose products are important for nucleocytoplasmic export of poly(A)+ RNA and encodes an essential nucleoporin. We report here the identification of RSS1 (Rat Seven Suppressor) as a high-copy extragenic suppressor of the rat7-1 temperature-sensitive allele. Rss1p encodes a novel essential protein of 538 amino acids, which contains an extended predicted coiled-coil domain and is located both at nuclear pore complexes (NPCs) and in the cytoplasm. RSS1 is the first reported high-copy extragenic suppressor of a mutant nucleoporin. Overexpression of Rss1p partially suppresses the defects in nucleocytoplasmic export of poly(A)+ RNA, rRNA synthesis and processing, and nucleolar morphology seen in rat7-1 cells shifted to the nonpermissive temperature of 37 degrees C and, thus, restores these processes to levels adequate for growth at a rate approximately one-half that of wild-type cells. After a shift to 37 degrees C, the mutant Rat7-1p/Nup159-1p is lost from the nuclear rim of rat7-1 cells and NPCs, which are clustered together in these cells grown under permissive conditions become substantially less clustered. Overexpression of Rss1p did not result in retention of the mutant Rat7-1p/Nup159-1p in NPCs, but it did result in partial maintenance of the NPC-clustering phenotype seen in mutant cells. Depletion of Rss1p by placing the RSS1 open reading frame (ORF) under control of the GAL1 promoter led to cessation of growth and nuclear accumulation of poly(A)+ RNA without affecting nuclear protein import or nuclear pore complex distribution, suggesting that RSS1 is directly involved in mRNA export. Because both rat7-1 cells and cells depleted for Rss1p are defective in mRNA export, our data are consistent with both gene products playing essential roles in the process of mRNA export and suggest that Rss1p overexpression suppresses the growth defect of rat7-1 cells at 37 degrees C by acting to maintain mRNA export.     

Edc3p and a glutamine/asparagine-rich domain of Lsm4p function in processing body assembly in Saccharomyces cerevisiae

Processing bodies (P-bodies) are cytoplasmic RNA granules that contain translationally repressed messenger ribonucleoproteins (mRNPs) and messenger RNA (mRNA) decay factors. The physical interactions that form the individual mRNPs within P-bodies and how those mRNPs assemble into larger P-bodies are unresolved. We identify direct protein interactions that could contribute to the formation of an mRNP complex that consists of core P-body components. Additionally, we demonstrate that the formation of P-bodies that are visible by light microscopy occurs either through Edc3p, which acts as a scaffold and cross-bridging protein, or via the "prionlike" domain in Lsm4p. Analysis of cells defective in P-body formation indicates that the concentration of translationally repressed mRNPs and decay factors into microscopically visible P-bodies is not necessary for basal control of translation repression and mRNA decay. These results suggest a stepwise model for P-body assembly with the initial formation of a core mRNA-protein complex that then aggregates through multiple specific mechanisms.     

Stress-dependent relocalization of translationally primed mRNPs to cytoplasmic granules that are kinetically and spatially distinct from P-bodies

Cytoplasmic RNA granules serve key functions in the control of messenger RNA (mRNA) fate in eukaryotic cells. For instance, in yeast, severe stress induces mRNA relocalization to sites of degradation or storage called processing bodies (P-bodies). In this study, we show that the translation repression associated with glucose starvation causes the key translational mediators of mRNA recognition, eIF4E, eIF4G, and Pab1p, to resediment away from ribosomal fractions. These mediators then accumulate in P-bodies and in previously unrecognized cytoplasmic bodies, which we define as EGP-bodies. Our kinetic studies highlight the fundamental difference between EGP- and P-bodies and reflect the complex dynamics surrounding reconfiguration of the mRNA pool under stress conditions. An absence of key mRNA decay factors from EGP-bodies points toward an mRNA storage function for these bodies. Overall, this study highlights new potential control points in both the regulation of mRNA fate and the global control of translation initiation.     

Physical and genetic interactions link the yeast protein Zds1p with mRNA nuclear export

Eukaryotic gene expression requires the export of mRNA from the nucleus to the cytoplasm. The DEAD box protein Dbp5p is an essential export factor conserved from yeast to man. A fraction of Dbp5p forms a complex with nucleoporins of the cytoplasmic filaments of the nuclear pore complex. Gfd1p was identified originally as a multicopy suppressor of the rat8-2 ts allele of DBP5. Here we reported that Dbp5p and Gfd1p interact with Zds1p, a protein previously identified as a multicopy suppressor in several yeast genetic screens. By using the two-hybrid system, we showed that Zds1p interacts in vivo with both Gfd1p and Dbp5p. In vitro binding experiments revealed that Gfd1p and Dbp5p bind directly to the C-terminal part of Zds1p. In addition, ZDS1 interacted genetically with mutant alleles of genes encoding key factors in mRNA export, including DBP5 and MEX67. Furthermore, deletion of ZDS1 or of both ZDS1 and the closely related ZDS2 exacerbated the poly(A)+ export defects shown by dbp5-2 and mex67-5 mutants. We proposed that Zds1p associates with the complex formed by Dbp5p, Gfd1p, and nucleoporins at the cytosolic fibrils of the nuclear pore complex and is required for optimal mRNA export.     

Elf1p,a member of the ABC class of ATPases,functions as a mRNA export factor in Schizosacchromyces pombe

Rae1p and Mex67p/Tap are conserved mRNA export factors. We have used synthetic lethal genetic screens in Schizosaccharomyces pombe to identify mutations in genes that are functionally linked to rae1 and mex67 in mRNA export. From these screens, we have isolated mutations in a putative S. pombe homologue of the Candida albicans elf1 gene. The elf1 of S. pombe is not an essential gene. When elf1 mutations are combined with rae1-167 mutation, growth and mRNA export is inhibited in the double mutants. This inhibition can be suppressed by the multicopy expression of mex67 suggesting that Mex67p can substitute for the loss of Elf1p function. Elf1p is a non-membrane member of the ATP-binding cassette (ABC) class of ATPase and the GFP-Elf1p fusion localizes to the cytoplasm. Elf1p, expressed and purified from Escherichia coli, binds and hydrolyzes ATP. A mutant Elf1p that carries a glycine to aspartic acid (G731D) mutation within the Walker A domain of the second ATP site retains the ATP binding but loses its ATPase activity in vitro. This mutant protein no longer functions in mRNA export. Taken together, our results show that Elf1p functions as a mRNA export factor along with Rae1p and Mex67p in S. pombe.     

Microtubule disruption stimulates P-body formation

Processing bodies (P-bodies) are subcellular ribonucleoprotein (RNP) granules that have been hypothesized to be sites of mRNA degradation, mRNA translational control, and/or mRNA storage. Importantly, P-bodies are conserved from yeast to mammals and contain a common set of evolutionarily conserved protein constituents. P-bodies are dynamic structures and their formation appears to fluctuate in correlation with alterations in mRNA metabolism. Despite these observations, little is understood about how P-body structures are formed within the cell. In this study, we demonstrate a relationship between P-bodies and microtubules in the budding yeast, Saccharomyces cerevisiae. First, we demonstrate that disruption of microtubules by treatment with the drug benomyl leads to aggregation of P-body components. Consistent with this finding, we also demonstrate that disruption of microtubules by a temperature-sensitive allele of the major alpha tubulin, TUB1 (tub1-724) stimulates P-body formation. Second, we find that the alpha-tubulin protein Tub1 colocalizes with P-bodies upon microtubule destabilization. Third, we determine that a putative tubulin tyrosine ligase, encoded by YBR094W, is a protein component of P-bodies, providing additional evidence for a physical connection between P-bodies and microtubules. Finally, we establish that P-bodies formed by microtubule destabilization fail to correlate with global changes in the stability of mRNA or in general mRNA translation. These findings demonstrate that the aggregation of P-body components is linked to the intracellular microtubule network, and, further, that P-bodies formed by disruption of microtubules aggregate independent of broad alterations in either mRNA decay or mRNA translation.     

Crp79p,like Mex67p,is an auxiliary mRNA export factor in Schizosaccharomyces pombe

The export of mRNA from the nucleus to the cytoplasm involves interactions of proteins with mRNA and the nuclear pore complex. We isolated Crp79p, a novel mRNA export factor from the same synthetic lethal screen that led to the identification of spMex67p in Schizosaccharomyces pombe. Crp79p is a 710-amino-acid-long protein that contains three RNA recognition motif domains in tandem and a distinct C-terminus. Fused to green fluorescent protein (GFP), Crp79p localizes to the cytoplasm. Like Mex67p, Crp79-GFP binds poly(A)(+) RNA in vivo, shuttles between the nucleus and the cytoplasm, and contains a nuclear export activity at the C-terminus that is Crm1p-independent. All of these properties are essential for Crp79p to promote mRNA export. Crp79p import into the nucleus depends on the Ran system. A domain of spMex67p previously identified as having a nuclear export activity can functionally substitute for the nuclear export activity at the C-terminus of Crp79p. Although both Crp79p and spMex67p function to export mRNA, Crp79p does not substitute for all of spMex67p functions and probably is not a functional homologue of spMex67p. We propose that Crp79p is a nonessential mRNA export carrier in S. pombe.     

Mitochondria associate with P-bodies and modulate microRNA-mediated RNA interference

P-bodies are cytoplasmic granules that are linked to mRNA decay, mRNA storage, and RNA interference (RNAi). They are known to interact with stress granules in stressed cells, and with late endosomes. Here, we report that P-bodies also interact with mitochondria, as previously described for P-body-related granules in germ cells. The interaction is dynamic, as a large majority of P-bodies contacts mitochondria at least once within a 3-min interval, and for about 18 s. This association requires an intact microtubule network. The depletion of P-bodies does not seem to affect mitochondria, nor the mitochondrial activity to be required for their contacts with P-bodies. However, inactivation of mitochondria leads to a strong decrease of miRNA-mediated RNAi efficiency, and to a lesser extent of siRNA-mediated RNAi. The defect occurs during the assembly of active RISC and is associated with a specific delocalization of endogeneous Ago2 from P-bodies. Our study reveals the possible involvement of RNAi defect in pathologies involving mitochondrial deficiencies.     

Analysis of P-body assembly in Saccharomyces cerevisiae

Recent experiments have defined cytoplasmic foci, referred to as processing bodies (P-bodies), that contain untranslating mRNAs in conjunction with proteins involved in translation repression and mRNA decapping and degradation. However, the order of protein assembly into P-bodies and the interactions that promote P-body assembly are unknown. To gain insight into how yeast P-bodies assemble, we examined the P-body accumulation of Dcp1p, Dcp2p, Edc3p, Dhh1p, Pat1p, Lsm1p, Xrn1p, Ccr4p, and Pop2p in deletion mutants lacking one or more P-body component. These experiments revealed that Dcp2p and Pat1p are required for recruitment of Dcp1p and of the Lsm1-7p complex to P-bodies, respectively. We also demonstrate that P-body assembly is redundant and no single known component of P-bodies is required for P-body assembly, although both Dcp2p and Pat1p contribute to P-body assembly. In addition, our results indicate that Pat1p can be a nuclear-cytoplasmic shuttling protein and acts early in P-body assembly. In contrast, the Lsm1-7p complex appears to primarily function in a rate limiting step after P-body assembly in triggering decapping. Taken together, these results provide insight both into the function of individual proteins involved in mRNA degradation and the mechanisms by which yeast P-bodies assemble.     

P-bodies and their functions during mRNA cell cycle: mini-review

P-bodies (processing bodies) are observed in different organisms such as yeast, Caenorhabditis elegans and mammals. A typical eukaryotic cell contains several types of spatially formed granules, such as P-bodies, stress granules and a variety of ribonucleoprotein bodies. These microdomains play important role in mRNA processing, including RNA interference, repression of translation and mRNA decay. The P-bodies components as well as stress granules may play an important role in host defense against viral infection. The complete set of P-bodies protein elements is still poor known. They contain conserved protein core limited to different organisms or to stress status of the cell. P-bodies are related also to some neuronal mRNA granules as well as to maternal RNA granules or male germ cell granules. In this mini-review, we focus on the structure of P-bodies and their function in the mRNA utilization and processing because of the high mRNA's dynamics between different cellular compartments and its key role in modulation of gene expression.     

Ge-1 is a central component of the mammalian cytoplasmic mRNA processing body

The mRNA processing body (P-body) is a cellular structure that regulates gene expression by degrading cytoplasmic mRNA. The objective of this study was to identify and characterize novel components of the mammalian P-body. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis have antibodies directed against this structure. Serum from one of these patients was used to identify a cDNA encoding Ge-1, a 1,401-amino-acid protein. Ge-1 contains an N-terminal WD 40 motif and C-terminal domains characterized by a repeating psi(X(2-3)) motif. Ge-1 co-localized with previously identified P-body components, including proteins involved in mRNA decapping (DCP1a and DCP2) and the autoantigen GW 182. The Ge-1 C-terminal domain was necessary and sufficient to target the protein to P-bodies. Following exposure of cells to oxidative stress, Ge-1-containing P-bodies were found adjacent to TIA-containing stress granules. During the recovery period, TIA returned to the nucleus while Ge-1-containing P-bodies localized to the perinuclear region. siRNA-mediated knock-down of Ge-1 resulted in loss of P-bodies containing Ge-1, DCP1a, and DCP2. In contrast, Ge-1-containing P-bodies persisted despite knock-down of DCP2. Taken together, the results of this study show that Ge-1 is a central component of P-bodies and suggest that Ge-1 may act prior to the 5(')-decapping step in mRNA degradation.     

Hepatitis C virus infection alters P-body composition but is independent of P-body granules

Processing bodies (P-bodies) are highly dynamic cytoplasmic granules conserved among eukaryotes. They are present under normal growth conditions and contain translationally repressed mRNAs together with proteins from the mRNA decay and microRNA (miRNA) machineries. We have previously shown that the core P-body components PatL1, LSm1, and DDX6 (Rck/p54) are required for hepatitis C virus (HCV) RNA replication; however, how HCV infection affects P-body granules and whether P-body granules per se influence the HCV life cycle remain unresolved issues. Here we show that HCV infection alters P-body composition by specifically changing the localization pattern of P-body components that are required for HCV replication. This effect was not related to an altered expression level of these components and could be reversed by inhibiting HCV replication with a polymerase inhibitor. Similar observations were obtained with a subgenomic replicon that supports only HCV translation and replication, indicating that these early steps of the HCV life cycle trigger the P-body alterations. Finally, P-body disruption by Rap55 depletion did not affect viral titers or HCV protein levels, demonstrating that the localization of PatL1, LSm1, and DDX6 in P-bodies is not required for their function on HCV. Thus, the HCV-induced changes on P-bodies are mechanistically linked to the function of specific P-body components in HCV RNA translation and replication; however, the formation of P-body granules is not required for HCV infection.