The translational regulator CPEB1 provides a link between dcp1 bodies and stress granules

A. Wilczynska*, C. Aigueperse, M. Kress, F. Dautry and D. Weil{ddagger}

CNRS UPR1983, Institut André Lwoff, 7 rue Guy Moquet, 94801 Villejuif CEDEX, France

{ddagger} Author for correspondence (e-mail: weil{at}vjf.cnrs.fr)

Accepted 21 December 2004


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The cytoplasmic polyadenylation element-binding protein (CPEB) has been characterized in Xenopus laevis as a translational regulator. During the early development, it behaves first as an inhibitor and later as an activator of translation. In mammals, its closest homologue is CPEB1 for which two isoforms, short and long, have been described. Here we describe an additional isoform with a different RNA recognition motif, which is differentially expressed in the brain and ovary. We show that all CPEB1 isoforms are found associated with two previously described cytoplasmic structures, stress granules and dcp1 bodies. This association requires the RNA binding ability of the protein, whereas the Aurora A phosphorylation site is dispensable. Interestingly, the rck/p54 DEAD box protein, which is known as a CPEB partner in Xenopus and clam, and as a component of dcp1 bodies in mammals, is also present in stress granules. Both stress granules and dcp1 bodies are involved in mRNA storage and/or degradation, although so far no link has been made between the two, in terms of neither morphology nor protein content. Here we show that transient CPEB1 expression induces the assembly of stress granules, which in turn recruit dcp1 bodies. This dynamic connection between the two structures sheds new light on the compartmentalization of mRNA metabolism in the cytoplasm.

Key words: CPEB, dcp1 body, GW body, Translation, Storage, Degradation


    Introduction
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 Introduction
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The translational control of mRNA is a way of regulating the production of proteins from pre-existing mRNAs within a short time scale. In higher eukaryotes, a well-known example is the regulation by cytoplasmic polyadenylation, which has been mainly described in Xenopus during oocyte activation and early development. As the mature oocytes have neither a transcriptional nor an mRNA degradation activity, the regulation of gene expression relies solely on this translational control. During oogenesis many mRNAs are hypoadenylated and stored in a dormant state. Following oocyte activation, they are readenylated under the control of the cytoplasmic polyadenylation element-binding protein (CPEB) and become competent for translation. Most of the known CPEB target mRNAs encode proteins that play a direct role in meiosis and mitosis, such as the mos kinase, cyclins and the Eg5 kinesin. This regulation is essential for the resumption of meiosis and the early embryonic development (Groisman et al., 2000Go). These observations have been extended to mammalian development and somatic cells, in particular to neurons, where CPEB target mRNAs are translated following synapse activation (Wells et al., 2000Go). In this case, the translational regulation is one of several control levels, as somatic cells continuously transcribe and degrade mRNAs. It may enable a rapid and local response of the cells to the stimulation of a single synapse. At the molecular level, CPEB activity is regulated by external stimuli such as progesterone in Xenopus oocytes and BDNF (brain derived neurotrophic factor) and NMDA (N-methyl-D-aspartate) in neurons, all of them leading to the activation of the Aurora A kinase. This kinase then directly phosphorylates CPEB protein (Wells et al., 2000Go). In the Xenopus oocyte, dephosphorylated CPEB is bound to maskin (Stebbins-Boaz et al., 1999Go). Maskin interacts with the translation initiation factor eIF4E present at the 5' extremity of the target mRNA, preventing the recruitment of eIF4G, and thus translation initiation. Following phosphorylation of CPEB, maskin releases eIF4E, whereas CPEB recruits the polyadenylation factor CPSF (cleavage and polyadenylation specificity factor) to the polyadenylation signal, leading to the subsequent polyadenylation of the mRNA (Mendez et al., 2000Go). This finally results in the resumption of translation initiation.

What is the fate of untranslated mRNAs within the cell? They can be stored in specific sites awaiting a signal able to trigger their translation. This is for instance the case of the neuronal mRNAs localized at synapses, which are translated following synapse activation. A well-documented example, {alpha}-CaMKII mRNA, is in fact a CPEB target (Aakalu et al., 2001Go; Wu et al., 1998Go). Alternatively, they can be routed to cytoplasmic structures that will direct them towards storage or degradation pathways. Two such structures have been described in fibroblasts: stress granules and dcp1 bodies.

In response to environmental stress such as oxidative conditions, UV irradiation or heat shock, global translation diminishes. In particular, the translational initiation factor eIF2-{alpha} is phosphorylated and therefore unable to reload GTP and recruit eIF5 to the preinitiation complex. As a result, abortive eIF2/eIF5-deficient 48S* complexes assemble on mRNAs and accumulate in cytoplasmic granules together with various mRNA binding proteins (Anderson and Kedersha, 2002Go). These so-called stress granules are thought to form as a result of intermingling of blocked mRNAs through the self-aggregation of the RNA binding proteins TIA-1 and TIAR. In the absence of stress, the overexpression of a phosphomimetic mutant of eIF2-{alpha} leads to the assembly of similar stress granules (Kedersha et al., 2002Go). The fact that stress granules are dynamic and reversible structures suggests that they could be sorting sites for the storage or degradation of untranslated mRNAs (Kedersha et al., 2002Go).

The machinery of the main mRNA degradation pathway, the 5'-3' mRNA decay, is concentrated in cytoplasmic foci both in mammals (Ingelfinger et al., 2002Go; van Dijk et al., 2002Go) and in yeast (Sheth and Parker, 2003Go), called dcp1 bodies and P-bodies, respectively. This includes the decapping enzymes dcp1 and dcp2, the 5'-3' exonuclease Xrn-1, proteins that stimulate decapping such as the LSm complex and the Dhh1p/rck/p54 protein, as well as an RNA binding protein of unknown function, GW182, initially identified as a human autoantigen (Eystathioy et al., 2002Go). Blocking the mRNA degradation pathway by genetic means in yeast (Sheth and Parker, 2003Go) or using siRNAs in mammals (Cougot et al., 2004Go) enhanced the accumulation of mRNAs in these foci, indicating that they are active sites of mRNA degradation rather than storage sites for these factors. Thus, mRNA degradation, at least in part, occurs in specific bodies and not diffusely throughout the cytoplasm as postulated previously.

Although one CPEB gene has been described in Xenopus laevis, four genes have been reported so far in mammals, named CPEB1 to CPEB4 (Gebauer and Richter, 1996Go; Kurihara et al., 2003Go; Theis et al., 2003Go; Welk et al., 2001Go), CPEB1 clearly being the closest homologue to the Xenopus gene. In addition, two isoforms, long and short, resulting from differential exons at the 5' extremity of the mRNA, have been described in humans (Welk et al., 2001Go). We have characterized a new CPEB1 isoform that differs in its first RNA recognition domain. Both previously described long and short isoforms as well as this new isoform are differentially expressed in the ovary and brain, two tissues where CPEB activity has been demonstrated (Groisman et al., 2000Go; Huang et al., 2002Go). All CPEB1 isoforms were recruited to two structures involved in mRNA storage and/or degradation: stress granules and dcp1 bodies. In addition, GFP-fused CPEB1 protein was able to assemble stress granules in the absence of stress. Finally, these granules in turn recruited components of dcp1 bodies, indicating a dynamic relationship between these two structures.


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Expression vectors
The human CPEB1-{Delta}5-long cDNA sequence from nucleotides 1 to 1791 (GenBank accession number BC050629/IMAGE:6047179) was inserted into the eukaryotic expression vector pEGFP-N1 (BD Biosciences Clontech, France), so that the complete CPEB1-{Delta}5-long open reading frame (ORF) was in-frame with the GFP ORF at the C-terminus. The CPEB1-{Delta}5-short expression vector was derived from this vector by restriction fragment exchange with the short cDNA (accession number H12139/IMAGE:48054). The CPEB1-long and -short plasmids were derived from the previous plasmids by restriction fragment exchange with the non-{Delta}5 cDNA (BG722145/IMAGE: 4830450). F314A, H545A, T172D and T172A point mutations were created in the CPEB1-long expression vector using the QuickChange XL site-directed mutagenesis kit (Stratagene, The Netherlands).

The Tet-regulated vector has been derived previously from the tTA expression vector (Dirks et al., 1994Go; Weil et al., 2000Go). The tTA/CPEB1-{Delta}5-long plasmid was created by inserting a restriction fragment containing the full ORF of the pEGFP/CPEB1-{Delta}5-long plasmid. The red fluorescent protein (RFP) cDNA was inserted upstream of the human rck/p54 cDNA in pCMV-SPORT6 vector (BC065007/IMAGE:6163439), so that the complete p54 ORF is in-frame with the RFP ORF at the N-terminus.

Cell culture
Epitheloid carcinoma HeLa cells and retinal pigment epithelial RPE-1 cells (BD Biosciences Clontech, France) were routinely maintained in DMEM and DMEM/F12, respectively, supplemented with 10% fetal calf serum. For stress induction, cells were treated with 0.5 mM arsenite (Sigma Aldrich, France) for 30 minutes, followed by recovery for 30 minutes in the absence of arsenite (Kedersha et al., 2002Go). Translation inhibition was achieved using 10 µg/ml cycloheximide (Roche Diagnostics, France) for 40 minutes or 100 µg/ml puromycin (Sigma) for 1 hour.

Transient transfections were performed with 1.5 µg plasmid DNA per 35 mm diameter dish by a standard calcium phosphate procedure (Sambrook et al., 1989Go). For stable transfection, HeLa rtTA HR5 (BD Biosciences Clontech, France) cells were co-transfected with 6 µg tTA/CPEB1-{Delta}5-long plasmid and 0.6 µg of hygromycin-resistant pY3 plasmid DNA (Blochlinger and Diggelmann, 1984Go) per 100 mm diameter dish using lipofectamine 2000 (Invitrogen, France). The selection of stable clones was achieved as described previously (Audibert et al., 2002Go). Cells were then routinely maintained in the presence of 100 µg/ml geneticin sulfate (Invitrogen, France) and 200 µg/ml hygromycin (Invitrogen, France). Induction of the Tet promoter was performed by addition of 1 µg/ml doxycycline to the culture medium.

RT-PCR
Reverse transcription reactions were performed with 1 µg total RNA using random primers and Mu-MLV reverse transcriptase (Invitrogen, France). One tenth of the reaction was used for PCR amplification. The position of the CPEB1 primers is indicated in Fig. 1A. The rrm5 (5'-ATGGCCAGGAGCTTCTGT-3') and rrm3 (5'-CGGCTGGACATCTTGAAA-3') primers were used for the amplification of all CPEB1 isoforms, lg5 (5'-GGGGTACCGCTGGGACAACCAAGGAAG-3') and ls3 (5'-GACTAGTCCAAGTCAGACCCAAGGG-3') for the amplification of the CPEB1-long isoform and sh5 (5'-GGGGTACCAGCGGGAAGCATCAGCAG-3') and ls3 for the amplification of the CPEB1-short isoform. After 37 cycles (94°C for 45 seconds, 60°C for 45 seconds and 72°C for 45 seconds), amplification products were separated on a 1.2% agarose gel and stained with ethidium bromide. Each primer set targeted two separate exons so that the amplification could be unambiguously attributed to reverse-transcribed mRNAs rather than residual genomic DNA. To distinguish between the {Delta}5 and non-{Delta}5 isoforms, the rrm5-rrm3 amplification products were digested with EcoNI restriction enzyme, separated on an 8% polyacrylamide gel and stained with ethidium bromide.



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Fig. 1. Alternative splicing of CPEB1 within the RRM1. (A) Schematic representation of hCPEB1 mRNAs. Human CPEB1-long (lg) and -short (sh) mRNAs are represented with the ORF in black including the two RRM domains in grey and the zinc finger domain (Zn). The short specific 5' UTR is hatched, with the optional intron indicated with a star. The primers and the EcoNI restriction site used are indicated by arrows and scissors, respectively. The black triangle indicates the position of the alternative 15 nucleotides. (B) Comparison of CPEB1 of various species. Partial RRM1 sequences from human, murine, Xenopus and Zebrafish EST and cDNA were aligned. The GT dinucleotide corresponding to a splice donor site is highlighted in grey. The encoded amino acids are indicated below the sequence. hsCPEB1-{Delta}5 is from GenBank accession number BX327041 (nucleotides 586-630), hsCPEB1 from AF329402 (nt. 1355-1414), mmCPEB1-{Delta}5 from BI144277 (nt. 395-439), mmCPEB1 from NM_007755 (nt. 1064-1123), xlCPEB from XLU14169(nt. 1114-1173) and drCPEB from AF076918 (nt. 1032-1091). (C) Position of the deletion with respect to RRM structure. {alpha} helices and ß sheets of the RRM are represented, the black triangle indicating the position of the alternative five amino acids. (D) Differential expression of CPEB1-long and -short in tissues and cell lines. CPEB1 mRNA from indicated samples was amplified by RT-PCR using common primers rrm5 and rrm3 (upper panel), CPEB1-long specific primers lg5 and ls3 (middle panel) or CPEB1-short specific primers sh5 and ls3 (lower panel), as illustrated in A. Amplification in the absence of RNA was used as a negative control (–). A 100 bp ladder was used as molecular weight marker (M). (E) Differential expression of the {Delta}5 isoform in tissues and cell lines. RNA was amplified by RT-PCR using rrm5 and rrm3 primers and digested with EcoN1, as illustrated in A. The {psi}X174 HaeIII digest was used as molecular weight marker (M).

 

Immunofluorescence
To raise anti-human CPEB1 antibodies, the human CPEB1-{Delta}5 cDNA sequence from nucleotides 310 to 2090 (GenBank accession number BC050629/IMAGE:6047179) was inserted into the prokaryotic expression vector pGEX6P3 (Amersham Pharmacia, France), so that the CPEB1 ORF was in-frame with the GST ORF at the N-terminus. This plasmid was transformed into Escherichia coli BLR (DE3) bacteria. The GST-tagged CPEB1 protein was purified on a preparative SDS-PAGE gel and injected into mice. Monoclonal antibodies recognizing the CPEB1 moiety of the fusion protein by immunofluorescence and western blotting were selected.

The anti-GW182 human index serum was a kind gift from Theophany Eystathoy (University of Calgary, Alberta, Canada), the anti-hDcp1 rabbit antibody from Bertrand Séraphin (Centre de Génétique Moléculaire, Gif, France) and the anti-eIF3 goat antibody from John Hershey (University of California, Davis, CA). The secondary antibodies conjugated to rhodamine and FITC were purchased from Jackson Immunoresearch Laboratories (Immunotech, France).

For immunofluorescence, cells were grown on glass coverslips and either fixed with 4% paraformaldehyde for 10 minutes and permeabilized with 0.5% Triton X-100, 50 mM NH4Cl for 15 minutes for anti-hDcp1 staining, or fixed in –20°C methanol for 3 minutes for anti-GW182 and anti-eIF3 staining. Cells were incubated with the primary antibody for 1 hour, rinsed with phosphate-buffered saline (PBS), incubated with the secondary antibody for 30 minutes, rinsed with PBS and stained with 0.12 µg/ml DAPI for 1 minute, all steps being performed at room temperature. Slides were mounted in Citifluor (Citifluor, UK) and observed on a Leica DMR microscope (Leica, Heidelberg, Germany) using a x63/1.32 oil-immersion objective. Photographs were taken using a Micromax CCD camera (Princeton Instruments). Confocal images were obtained on a Leica TCS-NT/SP inverted confocal laser-scanning microscope using an Apochromat x63/1.32 oil-immersion objective. Fluorescence signals were acquired in 0.16 µm optical sections.

Western blot analysis
Cells were scraped into PBS, resuspended in lysis buffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40) supplemented with complete protease inhibitor cocktail (Roche Diagnostics, France) and incubated on ice for 30 minutes. Soluble proteins were recovered after centrifugation at 15,000 g at 4°C for 10 minutes and quantified by the Bradford method (BioRad, France).

Proteins were separated on a 7.5% polyacrylamide SDS-PAGE gel along with a prestained protein ladder 10-180 kDa (MBI Fermentas, France) and transferred to a nitrocellulose membrane (Amersham Pharmacia, France). Non-specific protein binding sites were blocked by incubation in PBST (PBS with 0.1% Tween-20) containing 5% (w/v) non-fat dry milk for 1 hour at room temperature. The membrane was then incubated with the monoclonal antibody (1:5000 in PBST containing 5% non-fat dry milk) overnight at 4°C. After washing in PBST, the blot was incubated with horseradish peroxidase-conjugated goat anti-mouse IgG (1:10,000) (Immunotech Jackson, France) for 45 minutes at room temperature. After washing in PBST, immune complexes were detected using the SuperSignal® chemiluminescent substrate detection reagent (Perbio Science, France).


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 Materials and Methods
 Results
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 References
 
Variable CPEB1 isoforms in mammalian cells
Although only one CPEB isoform has been described in Xenopus laevis, two human CPEB1 mRNAs have been reported, encoding a long and a short CPEB1 isoform (Welk et al., 2001Go). The long form is 566 amino acids long and very similar to the Xenopus CPEB protein. The short form corresponds to a 68 amino acid N-terminal truncation of the long form, owing to the presence of alternative upstream exons (Fig. 1A).

We have identified in expressed sequence tag (EST) databanks a new CPEB1 mRNA containing a 15 nucleotide deletion in the open reading frame both in the human and mouse (Fig. 1B). Based on the human genome sequence, these 15 nucleotides are located precisely at the end of an exon. The deletion starts with a consensus GT dinucleotide, confirming that it results from an alternative splicing donor site. This variant mRNA encodes a CPEB1 protein with a five amino acid deletion (GNMPK) located in the first RNA recognition motif (RRM1) upstream of the third ß-sheet (Fig. 1C). We named it CPEB1-{Delta}5.

The expression of the long and short mRNAs and the presence of the {Delta}5 deletion were investigated by RT-PCR in murine brain and ovary tissues, as well as in the two human cell lines HeLa and RPE-1. The CPEB1 long form mRNA was present in the four samples, whereas the short form was detected only in the brain (Fig. 1D). In addition, the short form was amplified as two bands, probably corresponding to an optional intron of 211 nucleotides, which is present within the 5' UTR in several murine and human ESTs. To distinguish between the {Delta}5 and non-{Delta}5 isoforms, the region encompassing the alternative 15 nucleotides was amplified by RT-PCR, cleaved by the EcoNI restriction enzyme in order to reduce its size (Fig. 1A) and analysed on a polyacrylamide gel (Fig. 1E). Although both forms could be amplified from all samples, the non-{Delta}5 form was most abundant in the ovary, and the {Delta}5 form in the brain and cell lines. Therefore, both the long/short and the {Delta}5 alternative splicing were tissue specific.

CPEB1 localization in cytoplasmic foci
In order to study the intracellular localization of the CPEB1 protein, monoclonal antibodies were raised against the human CPEB1-{Delta}5 protein fused to GST and produced in E. coli. Their specificity was demonstrated by western blotting using extracts of HeLa cells transfected with CPEB1 expression vectors (Fig. 2A). As an example, the 2B7 antibody identified both the human long and mouse short CPEB1-{Delta}5 proteins at sizes consistent with their predicted molecular weight of 62 and 53 kDa, respectively. This antibody was then used to determine the intracellular localization of endogenous CPEB1 protein in HeLa cells by immunofluorescence (Fig. 2B). The staining was weak and concentrated in a few discrete cytoplasmic foci.



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Fig. 2. CPEB1 localization in HeLa cells. (A) Western blot assay. The anti-CPEB1 monoclonal antibody 2B7 was used for western blotting of proteins from HeLa cells expressing the murine CPEB1-{Delta}5-short or the human CPEB1-{Delta}5-long isoform. Untransfected HeLa cells were used as a control. (B) Immunofluorescence of untransfected HeLa cells. Cells were fixed, stained with anti-CPEB1 antibody and observed by fluorescence microscopy. Bar, 8 µm.

 

The specificity of these immunofluorescence data was confirmed using GFP-tagged CPEB1 protein. An open reading frame encoding human CPEB1-{Delta}5-long fused to GFP at the C-terminus, was introduced into a plasmid under the control of a CMV promoter and transiently transfected into HeLa cells. In about two thirds of the cells, CPEB1-{Delta}5 exhibited a cytoplasmic diffuse localization with a few discrete enriched foci (Fig. 3A), as observed with the anti-CPEB1 antibody in untransfected HeLa cells. In the remaining cells, the protein was concentrated in larger granules (Fig. 3B).



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Fig. 3. Localization of GFP-tagged CPEB1 in HeLa cells. (A,B) Localization of CPEB1 after transient transfection. HeLa cells were transiently transfected with an expression vector for GFP-tagged human CPEB1-{Delta}5-long. After 24 hours, cells were fixed, stained with DAPI and observed by fluorescence microscopy. CPEB fluorescence and DAPI staining are on the left and right, respectively. Cells harbouring small cytoplasmic foci (arrows) and larger granules are illustrated in A and B, respectively. (C,D) Localization of CPEB1 after stable transfection. HeLa/CPEB1-lg cells were induced for 16 hours with doxycycline, fixed and stained with DAPI (C) or observed live (D). The arrows indicate small cytoplasmic foci. Bar, 8 µm.

 

The same open reading frame was also inserted into a plasmid under the control of a cytomegalovirus/tet promoter and transfected into the HeLa rtTA HR5 cell line to allow for inducible expression. Stable transfectants were selected and a clone with a high level of induction by doxycycline, called HeLa/CPEB1-lg, was chosen for further study. In every induced cell, the intracellular localization of the GFP-tagged CPEB1-{Delta}5 protein consisted of a diffuse cytoplasmic staining as well as a few discrete cytoplasmic foci (Fig. 3C). This localization in foci was also observed in living cells (Fig. 3D). However, the larger granules previously observed in transient transfection were absent.

Induction of stress granules by CPEB
We first analysed the large CPEB1 granules observed following transient transfection. Their morphology was similar to the previously described cytoplasmic stress granules (Kedersha et al., 2002Go). These granules can be induced by stress such as arsenite, UV or heat shock, as well as by expression of a phosphomimetic mutant of the translation initiation factor eIF2-{alpha}. They contain many translation initiation factors, in particular eIF3, mRNAs and some mRNA binding proteins.

HeLa cells were transiently transfected with the GFP-tagged CPEB1-{Delta}5-long expression vector and analysed with an anti-eIF3 antibody. The localization of eIF3 was diffuse in untransfected cells as well as in those where the CPEB1 protein was diffuse. However, when cells harboured larger CPEB1 granules, eIF3 was enriched in these granules (Fig. 4A). In addition, transfected cells were stressed with arsenite in order to induce stress granules. As previously described (Kedersha et al., 2002Go), all cells harboured eIF3-containing granules. Endogenous CPEB1 became undetectable using our antibodies (see later). However, the transfected CPEB1 protein was systematically enriched in these granules (Fig. 4B). The same result was obtained when the stably transfected HeLa/CPEB1-lg cells were induced and stressed with arsenite (data not shown).



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Fig. 4. Large CPEB1 granules are stress granules. HeLa cells were transiently transfected with an expression vector for GFP-tagged human CPEB1-{Delta}5-long. After 24 hours, cells were directly fixed (A) or stressed with arsenite for 30 minutes and fixed (B), then stained with anti-eIF3 antibodies (red) and observed by fluorescence microscopy.

 

In conclusion, CPEB1 protein was recruited into stress granules during stress, and its transient overexpression in unstressed cells was able to induce the formation of stress granule-like structures,as does a phosphomimetic mutant of eIF2-{alpha}.

Cytoplasmic CPEB1 small foci are dependent on translation
We then turned to the smaller CPEB1 cytoplasmic foci. As CPEB1 protein regulates polyadenylation and translation, we investigated whether the presence of these foci was dependent upon active translation. HeLa/CPEB1-lg cells were treated with two translation inhibitors, cycloheximide and puromycin. After 1 hour neither the cycloheximide nor the puromycin had notably decreased the abundance of the diffuse CPEB1 protein. However, there was a striking difference in the small cytoplasmic foci (Fig. 5A). They disappeared in the presence of cycloheximide, whereas puromycin enhanced their intensity and increased their number. The number of cells with more than five foci rose from 3% to 37% and the number of cells without any foci decreased from 40% to 10% (Fig. 5B).



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Fig. 5. Small CPEB1 foci depend on translation. (A) HeLa/CPEB1-lg cells were induced for 16 hours with doxycycline, treated with cycloheximide (CHX) for 40 minutes or puromycin for 1 hour, then fixed and observed by fluorescence microscopy. (B) The number of CPEB1 foci per cell was counted in 150 control, cycloheximide- and puromycin-treated cells.

 

These results indicated that CPEB1 foci are dynamic structures. The mechanism of action of the two translation inhibitors is different: puromycin leads to the disruption of polysomes and to the release of both the nascent peptides and the mRNAs, whereas cycloheximide traps arrested mRNA on polysomes. As the number of CPEB1 foci increased after puromycin treatment, they could not correspond to structures involved in active translation. In contrast, the effect of cycloheximide indicated that these foci depended on the ability of mRNAs to leave polysomes at the issue of translation.

Cytoplasmic CPEB1 foci are dcp1 bodies
These characteristics were reminiscent of the properties of dcp1 bodies in mammals (Cougot et al., 2004Go) and P-bodies in yeast (Sheth and Parker, 2003Go), two cytoplasmic structures involved in mRNA degradation. They have been shown to disappear following cycloheximide treatment and to be enhanced following the disruption of the mRNA degradation pathway. In mammals, the decapping enzyme dcp1 (Ingelfinger et al., 2002Go; van Dijk et al., 2002Go) and the mRNA binding protein GW182 (Eystathioy et al., 2002Go) specifically accumulate in dcp1 bodies.

To determine whether the CPEB1 foci were related to dcp1 bodies, we induced HeLa/CPEB1-lg cells with doxycycline and performed immunofluorescence experiments with either anti-dcp1 or anti-GW182 antibodies. The CPEB1 cytoplasmic foci fully colocalized with both dcp1 (Fig. 6A) and GW182 foci (Fig. 6B). The same results were obtained when the unfused human CPEB1-{Delta}5-long expression vector was transiently transfected into HeLa cells and stained with the anti-CPEB1 antibody (Fig. 6C), ensuring that the colocalization with dcp1 and GW182 was not due to the GFP moiety of the recombinant CPEB1 expressed in HeLa/CPEB1-lg cells. Finally, the endogenous CPEB1 protein detected with the anti-CPEB1 antibody also colocalized with the dcp1 protein in untransfected HeLa cells (Fig. 6D). In conclusion, the small CPEB1 foci corresponded to the previously described dcp1 bodies.



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Fig. 6. Small CPEB1 foci are dcp1/GW bodies. HeLa/CPEB1-lg cells were induced for 16 hours with doxycycline, fixed and stained with anti-dcp1 (A) or anti-GW182 (B) antibodies (red). HeLa cells transiently transfected with an expression vector for untagged human CPEB1-{Delta}5-long (C) and untransfected HeLa cells (D) were fixed and stained with anti-dcp1 (red) and anti-CPEB1 (green) antibodies. DAPI staining is blue.

 

Functional RRMs are required for CPEB1 targeting to both stress granules and dcp1 bodies
The existence of several mammalian CPEB1 isoforms, CPEB1-long and -short, {Delta}5 or not, raised the possibility of multiple functions for CPEB1, possibly related to distinct localizations. To address this question, the four isoforms were introduced as GFP fusions into expression vectors and transiently transfected in HeLa cells. All of them had a similar cytoplasmic localization, with presence of the protein in either stress granules (Fig. 7A) or dcp1 bodies (Fig. 7B), as confirmed with anti-eIF3 and anti-dcp1 antibodies, respectively. Therefore, neither the first 68 amino acids nor the GNMPK alternative motif were involved in the stress granule assembly or in the localization in dcp1 bodies.



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Fig. 7. Stress granule assembly and dcp1 bodies depend on the functionality of the mRNA binding domain. (A) The four CPEB1 isoforms can assemble stress granules. HeLa cells were transiently transfected with expression vectors for the indicated GFP-tagged CPEB1 isoforms. After 24 hours, cells were fixed, stained with anti-eIF3 antibodies (red) and observed by confocal microscopy. The figure illustrates only cells harbouring stress granules. (B) All CPEB1 isoforms can colocalize with dcp1 bodies. Cells obtained as in A were stained with anti-dcp1 (red) and observed by confocal microscopy. The figure illustrates only cells harbouring small CPEB1 foci. (C) A C-terminal truncation of CPEB1 abrogates the colocalization with dcp1 bodies. HeLa cells were transiently transfected with an expression vector for an RRM- and Zn finger-deleted CPEB1-long. After 24 hours, cells were fixed and stained with anti-dcp1 antibodies (red). (D) A C-terminal truncation of CPEB1 abrogates the localization in stress granules. HeLa cells transfected as in C were stressed with arsenite for 30 minutes, fixed and stained with anti-eIF3 antibodies (red). (E) H545A and F314A point mutations abrogate the localization of CPEB1 in dcp1 bodies. HeLa cells were transfected with an expression vector for GFP-tagged CPEB1-long-H545A or -F314A and stressed as in D. Cells were stained with anti-dcp1 antibodies (red).

 

As a first approach to determine the functional domain involved in these localizations, two large deletions were created in the GFP-tagged CPEB1-long expression vector, one removing the amino acids 1 to 237 at the N-terminus, and the other amino acids 330 to 566 at the C-terminus of the protein. The former deletion encompassed the Aurora A phosphorylation site and the PEST region, whereas the latter removed the RRMs and Zn finger so that the protein could not bind RNA (Hake et al., 1998Go). The N-terminal truncated protein was identical to the full-length protein in its capacity to assemble stress granules. It was also recruited into dcp1 bodies, although with less efficiency than the full-length protein (data not shown). In contrast, neither stress granules nor smaller CPEB1 foci were observed with the C-terminal truncated mutant, despite the presence of proper dcp1 foci within the cells (Fig. 7C). Moreover, this mutant was not recruited into stress granules following arsenite treatment (Fig. 7D).

To investigate in more detail the role of Aurora A phosphorylation and mRNA binding, we introduced a set of specific point mutations. The Thr172 targeted by Aurora A was mutated either to Asp, known as a phosphomimetic mutation, or to Ala, a non-phosphorylatable amino acid. None of these mutations affected the localization of CPEB1 in the cells (data not shown), confirming that Aurora A phosphorylation is not involved here. For inhibition of mRNA binding, we created two independent mutations. One was a His545 to Ala mutation within the zinc finger, previously shown to inactivate the mRNA binding domain of Xenopus CPEB (Hake et al., 1998Go). The other is a Phe314 to Ala mutation in the rnp2 motif of RRM1, known to abolish mRNA binding in RRM-containing proteins (Jessen et al., 1991Go). Both behaved like the C-terminal truncated mutant, harbouring neither dcp1 bodies nor stress granules (Fig. 7E).

Therefore both stress granule assembly and localization in dcp1 bodies required a functional mRNA binding domain, whereas the Aurora A phosphorylation site and the PEST domain were dispensable.

Stress granules recruit dcp1 bodies
The fact that the CPEB1 protein could both accumulate in dcp1 bodies and assemble stress granules, two structures involved in mRNA storage and/or degradation, led us to investigate the relationship between the two. HeLa cells were transiently transfected with GFP-tagged CPEB1-{Delta}5-short to induce stress granules and analysed for dcp1 localization after 20 hours. Several patterns were observed in the stress granule-containing cells. In 70% of cells, the punctuated dcp1 bodies had disappeared and dcp1 protein was present in the stress granules (Fig. 8A). In 10% of cells, punctuated dcp1 bodies coexisted with and were distinct from stress granules (Fig. 8B). In the remaining 20%, dcp1 bodies were distinct but closely associated with stress granules. There were often two to three bodies contacting one stress granule (Fig. 8C). The presence of GW182 in these cells was also analysed. Similar to dcp1, GW182 bodies had disappeared and the protein accumulated in stress granules (Fig. 8D). Therefore, stress granules induced by CPEB1 recruited two components of dcp1 bodies. Although the two proteins may join the stress granules separately, the image of dcp1 bodies surrounding stress granules suggested rather that dcp1 bodies were recruited as a whole by stress granules.



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Fig. 8. Stress granules induced by CPEB1, but not by arsenite, recruit components of dcp1 bodies. (A-D) CPEB1-induced stress granules recruit dcp1 and GW182. HeLa cells were transiently transfected with an expression vector for GFP-tagged CPEB1-{Delta}5-short. After 20 hours, cells were fixed and stained with anti-dcp1 (A-C) or anti-GW182 (D) antibodies (red). Cells were observed by confocal microscopy. As several patterns were observed, three cells are shown in A, B and C, which are representative of 70%, 10% and 20% of the stress granule-containing cells, respectively. In C, two stress granules surrounded with several dcp1 bodies have been enlarged for clearer visualization. (E) Arsenite increases the number of dcp1 bodies. HeLa/CPEB1-lg cells were induced for 16 hours with doxycycline, stressed with arsenite (as) for 30 minutes, or stressed and cultured further in the absence of arsenite for 1 hour (as +1 hour). After fixation, cells were stained with anti-dcp1 antibodies and observed by fluorescence microscopy. The graph presents the number of dcp1 bodies per cell, counted in 180 cells. (F) Recruitment of dcp1 in CPEB1-induced stress granules increases with time. HeLa cells transfected as in A were fixed at various time after transfection and stained with anti-dcp1 antibodies. CPEB1-expressing cells were counted for the presence of stress granules (SG) and the presence of dcp1 in these stress granules. Stress granule-containing cells are plotted as a percentage of CPEB1-expressing cells, whereas cells with dcp1 in stress granules are plotted as a percentage of stress granule-containing cells.

 

It was recently reported that dcp1 is absent from stress granules induced with arsenite in HEK293 cells (Cougot et al., 2004Go). We analysed dcp1 localization in our HeLa/CPEB1-lg cells treated with arsenite for 30 minutes. As in HEK293 cells, dcp1 was not recruited into newly assembled stress granules (data not shown). After recovery for 1 hour 45 minutes in the absence of arsenite, dcp1 was still not recruited and after 2 hours 30 minutes, stress granules had disappeared. It was interesting that the number of dcp1 bodies strongly increased in response to arsenite (Fig. 8E), which is consistent with its inhibitory effect on translation. It also suggests that, in this case, many repressed mRNAs are still targeted to dcp1 bodies rather than stress granules.

The fact that dcp1 joins CPEB1-induced but not arsenite-induced stress granules may be due to a difference in the timing of the observation. We therefore analysed dcp1 localization during shorter time of transfection. GFP-tagged CPEB1 expression became visible after 8 hours of transfection and cells were observed up to 24 hours. Among transfected cells, the number of stress granule-containing cells increased with time (Fig. 8F). In addition, among the latter, the number of cells where dcp1 had joined stress granules also increased with time (Fig. 8F). This indicated that stress granules take time to assemble and need further time to recruit dcp1 bodies.

The rck/p54 protein is colocalized with CPEB1 in both stress granules and dcp1 bodies
The DEAD box helicase rck/p54 is known as a CPEB partner in Xenopus and clam (Minshall and Standart, 2004Go; Minshall et al., 2001Go). It is also a component of P-bodies (Sheth and Parker, 2003Go) and dcp1 bodies (Cougot et al., 2004Go). We therefore analysed its localization following CPEB1 transfection and arsenite treatment.

An expression vector for RFP-tagged human p54 was constructed and transfected into HeLa cells. As expected, the fusion protein concentrated in dcp1 bodies, as confirmed by dcp1 colocalization (Fig. 9A), and did not trigger stress granule assembly. When RFP-tagged p54 was cotransfected with GFP-tagged CPEB1-long the protein systematically colocalized with CPEB1, either in dcp1 bodies (Fig. 9B, upper panel) or in CPEB1-induced stress granules (Fig. 9B, lower panel). Finally, p54 was transfected alone for 20 hours and stress granules were induced with arsenite. In half of the cells, p54 was found in stress granules, as confirmed by anti-eIF3 antibodies (Fig. 9C, middle panel). In the remaining cells, it was present in punctuated dcp1 bodies. These bodies were distinct from but often associated with stress granules (Fig. 9C, lower panel). We analysed endogenous CPEB1 in these cells using our anti-CPEB1 antibody. In contrast to untransfected cells, the endogenous protein was now detectable in the stress granules (Fig. 9D).



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Fig. 9. Human p54 colocalizes with CPEB1 in both dcp1 and stress granules. (A) p54 is found in dcp1 bodies. HeLa cells were transiently transfected with an expression vector for RFP-tagged p54. After 20 hours, cells were fixed, stained with anti-dcp1 antibodies (green), and observed by confocal microscopy. (B) p54 colocalizes with CPEB1. HeLa cells were cotransfected with RFP-tagged p54 and GFP-tagged CPEB1-long. After 20 hours, cells were fixed and observed by confocal microscopy. Cells without (upper panel) and with stress granules (lower panel) are presented. (C,D) p54 is recruited with endogenous CPEB1 in stress granules induced with arsenite. HeLa cells were transiently transfected with an expression vector for RFP-tagged p54. After 20 hours, cells were stressed with arsenite for 30 minutes, incubated in the absence of arsenite for 1 hour and fixed. After staining with anti-eIF3 (C) or anti-CPEB1 antibodies (D) (green), cells were observed by confocal microscopy. In C, two p54 foci contacting stress granules have been enlarged for better visualization.

 

Therefore, p54 is colocalized with CPEB1 in dcp1 bodies, as well as in stress granules, whether induced by CPEB1 or arsenite. For comparison, dcp1 is recruited in only 70% of the CPEB1-induced stress granules and is absent in arsenite-induced stress granules.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Two CPEB1 isoforms have been described in mammals, CPEB1-long and -short, resulting from the alternative splicing of the first coding exon. Here we show the existence of a second alternative splicing, which leads to a deletion of five amino acids within the RRM1. This new isoform, named CPEB1-{Delta}5, was also found among zebrafish ESTs. Both the long/short and the {Delta}5 alternative splicing were differentially regulated in mouse brain and ovary. The long form was common to both tissues, whereas the short form was exclusively found in the brain. In addition, the non-{Delta}5 and the {Delta}5 forms were predominant in the ovary and in the brain, respectively. This was in agreement with the single CPEB isoform cloned in Xenopus oocytes, which is long and non-{Delta}5. In two human cell lines, HeLa and RPE-1, only the long form was detected and predominantly the {Delta}5 form, as in the mouse brain.

The five amino acids that are deleted in the CPEB1-{Delta}5 isoform are located within the RRM1, at the C-terminal extremity of the ß2-ß3 loop. It is noteworthy that, for U1A, sx1 and nucleolin, amino acids at this position have been shown to interact directly with RNA of the RRM (Allain et al., 2000Go; Handa et al., 1999Go). A deletion in this region could therefore affect the RNA binding properties of CPEB1, at the affinity or specificity level. Its differential expression in ovary and brain could correspond to the existence of different targets in these tissues, for instance mitosis-related and signal transduction-related mRNAs, as postulated for oocyte and neurons, respectively (Wells et al., 2000Go).

The intracellular localization of the CPEB1 protein was investigated in HeLa cells using a monoclonal antibody developed in the laboratory. The protein was detected in small cytoplasmic foci. To distinguish between the four CPEB1 isoforms, expression vectors for GFP-tagged CPEB1 and CPEB1-{Delta}5, -long and -short, were transiently transfected in HeLa cells. The four isoforms had a similar diffuse cytoplasmic localization, with either small, dispersed foci or much larger granules that were irregular in size. In stably transfected HeLa cells, only the first pattern was observed.

The large CPEB1 granules observed in transiently transfected HeLa cells correspond to so-called stress granules. These granules are formed in response to stress such as arsenite, UV and heat shock, but can also be induced by the expression of a phosphomimetic mutant of eIF2-{alpha} (Kedersha et al., 2002Go). Their assembly results from the accumulation of abortive translation preinitiation complexes, which are deficient in eIF2/eIF5 (Anderson and Kedersha, 2002Go). As for the mutated eIF2-{alpha}, the overexpression of GFP-tagged CPEB1 induced the assembly of granules that contain the translation initiation factor eIF3, a marker of stress granules (Kedersha et al., 2002Go). Conversely, stress granules induced by arsenite (Fig. 4), UV or heat shock (data not shown) recruit CPEB1 protein. In Xenopus oocytes, CPEB1 represses translation initiation of its target mRNAs by preventing the binding of eIF4G to eIF4E. In mammalian cells, an excess of CPEB1 could repress translation, leading to the assembly of defective initiation complexes, which would accumulate in stress granules, enabling the storage or the degradation of blocked mRNAs. Stress granule inducers reported to date are: a phosphomimetic mutant of eIF2-{alpha}, a factor which belongs to the translation initiation complex (Kedersha et al., 2002Go), and two RNA binding proteins, the Fragile X Mental Retardation Protein (FMRP), which represses translation (Mazroui et al., 2002Go) and the Ras-GAP SH3 domain-binding protein (G3BP), which has an endoribonuclease activity (Tourriere et al., 2003Go). Thus CPEB1 is the second example of a translational regulator that can induce stress granules and the efficiency of this induction could indicate that CPEB1 has many more target mRNAs than anticipated.

The small CPEB1 foci observed in stably and transiently CPEB1-transfected cells correspond to previously reported dcp1 bodies. These cytoplasmic bodies have been described in both yeast and mammalian cells. They contain factors participating in the 5'-3' mRNA decay pathway, including the decapping enzymes dcp1 and dcp2, the exonuclease Xrn1, the Sm-like proteins LSm1 to 7, the deadenylase hCcr4 and the helicase dhh1/rck/p54 (Cougot et al., 2004Go; Ingelfinger et al., 2002Go; Sheth and Parker, 2003Go; van Dijk et al., 2002Go). They also contain an RNA binding protein of unknown function, GW182, initially identified as a human autoantigen (Eystathioy et al., 2002Go; Eystathioy et al., 2003Go). CPEB1 foci were indistinguishable from dcp1 or GW182 foci.

In yeast, these foci have been shown to be mRNA degradation sites rather than storage sites (Sheth and Parker, 2003Go). In particular, mutations that block decapping or 5'-3' mRNA degradation increase the number and size of these foci, whereas translation inhibition by cycloheximide, which traps mRNAs on polysomes, causes their disappearance. Here, we provide similar functional data in mammalian cells. On the one hand, mRNA release from polysomes by puromycin treatment led to a strong increase in the number of foci. On the other hand, blocking mRNAs on polysomes by cycloheximide treatment led to the disappearance of CPEB1 foci, as in yeast. These observations are consistent with a recent publication reporting that reducing 5'-3' mRNA degradation by Xrn-1 silencing leads to an increased number of dcp2 bodies, whereas cycloheximide treatment diminishes their number (Cougot et al., 2004Go). Together, these data demonstrate that these foci are dynamic and correspond to active sites of mRNA degradation.

The CPEB1 protein can be seen as both a translational activator when phosphorylated and recruiting the polyadenylation complex, and a translational inhibitor when dephosphorylated and bound to a protein such as maskin, which interferes with eIF4E function in translation initiation (Stebbins-Boaz et al., 1999Go). We postulate that the presence of CPEB1 in dcp1 bodies is probably related to its function as a translational inhibitor. Accordingly, the Aurora A phosphorylation site, which controls CPEB activity as a translational activator, is not required for this localization. The capacity of the four CPEB1 isoforms to associate with these bodies was indistinguishable and therefore did not suggest any difference in their respective inhibitor activity. The presence of CPEB1 in the bodies did require a functional mRNA binding domain, indicating that the protein is bound to its target mRNAs within them. Most proteins documented so far in dcp1 bodies are proteins directly involved in mRNA degradation. An RNA binding protein involved in translational repression such as CPEB1 could trigger the recruitment of its target mRNAs to dcp1 bodies and, as a consequence, be dragged into them. Along these lines, an MS2-GFP tag, carried along by an mRNA containing MS2 binding sites, was visualized in P-bodies, provided that the mRNA was enriched in that location by inhibition of either mRNA translation or mRNA degradation (Sheth and Parker, 2003Go). However, it may not be so simple, as the yeast Puf3p protein, albeit a regulator of mRNA degradation, is not enriched in dcp1 bodies (Sheth and Parker, 2003Go). Alternatively, the accumulation of CPEB1 in these bodies could reveal an active role for the protein in mRNA degradation. In this respect, it is of interest that mammalian rck/p54 accumulates in dcp1 bodies. Its homologues in the Xenopus and clam (Xp54 and p47, respectively) are known to be CPEB partners and translational repressors during early development (Minshall and Standart, 2004Go; Minshall et al., 2001Go). Its homologue in yeast, Dhh-1, binds to dcp1 and is required for efficient decapping (Coller et al., 2001Go; Fischer and Weis, 2002Go). This raises the possibility that CPEB1 is bound to rck/p54 within the dcp1 body and plays an active role in decapping.

Finally, our results illustrate a link between stress granules and dcp1 bodies, as schematically proposed in Fig. 10. It has been recently reported that stress granules newly assembled in arsenite-treated cells and dcp1 bodies do not overlap, although they are occasionally close together (Cougot et al., 2004Go). These authors concluded that the two structures were distinct. Similarly, when treating cells with arsenite, we observed distinct stress granules and dcp1 bodies up to the time at which stress granules disappear, i.e. 2.5 hours. In contrast, when stress granules were induced by CPEB1 expression, the typical dcp1 bodies had disappeared in most cells and the dcp1 and GW182 proteins previously present in these bodies filled the stress granules. This difference between arsenite-induced and CPEB1-induced stress granules may result from a difference in the timing of the observation. The former were observed shortly after assembly, whereas the latter were observed after 20 hours, allowing more time to recruit further factors. Supporting that hypothesis, we found that the recruitment of dcp1 into stress granules strongly increases between 8 hours and 20 hours after transfection. Interestingly, intermediate situations were observed, where the dcp1 bodies had not yet disappeared, but were adjacent to the stress granules. There was often more than one dcp1 body per stress granule, as if stress granules would progressively recruit and fuse with dcp1 bodies. When p54 was overexpressed in the cells, it was recruited in CPEB1-induced stress granules, like dcp1 and GW182. Moreover, it could also be recruited in arsenite-induced stress granules, along with endogenous CPEB1. Again, when p54 was still in dcp1 bodies, these bodies were most often adjacent to stress granules. Therefore, the composition of arsenite-induced stress granules is highly dependent on the cellular content. A high expression of p54 seems to accelerate the recruitment of dcp1 bodies. This communication between stress granules and dcp1 bodies is the first direct evidence for the postulated role of stress granules, which is to sort mRNAs for storage or degradation.



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Fig. 10. Proposed model for the link between stress granules and mRNA degradation bodies. Translation block in response to stress or translational repression by factors such as FMRP, mutated eIF2-{alpha} or GFP-CPEB, leads to the storage of mRNAs in stress granules. These granules can either revert if the stress disappears or progressively recruit dcp1 bodies to degrade mRNAs. In contrast, translation inhibitors enabling the release of mRNAs, like puromycin, lead directly to the default mRNA degradation pathway which takes place within the dcp1 bodies. However, translation inhibitors that trap arrested mRNAs on polysomes, such as cycloheximide (CHX), prevent them from joining the dcp1 bodies.

 


    Acknowledgments
 
We thank Michèle Huesca for raising monoclonal antibodies, Marie-Annick Harper for technical help and Gérard Pierron for reading of the manuscript. A.W. was supported by a Marie Curie Fellowship (5th PCRD, contract QLGA-1999-50406) and a FEBS Collaborative Experimental Scholarship for Central and Eastern Europe. This work was supported by the Centre National de la Recherche Scientifique and the Marie Curie Fellowship.


    Footnotes
 
* Present address: Molecular Biology Department, Cancer Center-Institute, ul. Roentgena 5, 02-781 Warsaw, Poland Back


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 Discussion
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