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First published online February 23, 2005
doi: 10.1242/10.1242/jcs.01692

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

A. Wilczynska^*, C. Aigueperse, M. Kress, F. Dautry and D. Weil

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

View larger version (49K): [in a new window]   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. hsCPEB1-5 is from GenBank accession number BX327041 (nucleotides 586-630), hsCPEB1 from AF329402 (nt. 1355-1414), mmCPEB1-5 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. 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 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 X174 HaeIII digest was used as molecular weight marker (M).

View larger version (48K): [in a new window]   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 CPEB1-5-short or the human CPEB1-5-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.

View larger version (69K): [in a new window]   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 CPEB1-5-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.

View larger version (61K): [in a new window]   Fig. 4. Large CPEB1 granules are stress granules. HeLa cells were transiently transfected with an expression vector for GFP-tagged human CPEB1-5-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.

View larger version (36K): [in a new window]   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.

View larger version (67K): [in a new window]   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 CPEB1-5-long (C) and untransfected HeLa cells (D) were fixed and stained with anti-dcp1 (red) and anti-CPEB1 (green) antibodies. DAPI staining is blue.

View larger version (79K): [in a new window]   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).

View larger version (26K): [in a new window]   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 CPEB1-5-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.

View larger version (57K): [in a new window]   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.

View larger version (14K): [in a new window]   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- 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.

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