UVC-Induced Stress Granules in Mammalian Cells

<div><p>Stress granules (SGs) are well characterized cytoplasmic RNA bodies that form under various stress conditions. We have observed that exposure of mammalian cells in culture to low doses of UVC induces the formation of discrete cytoplasmic RNA granules that were detected by immunofluorescence staining using antibodies to RNA-binding proteins. UVC-induced cytoplasmic granules are not Processing Bodies (P-bodies) and are <i>bone fide</i> SGs as they contain TIA-1, TIA-1/R, Caprin1, FMRP, G3BP1, PABP1, well known markers, and mRNA. Concomitant with the accumulation of the granules in the cytoplasm, cells enter a quiescent state, as they are arrested in G₁ phase of the cell cycle in order to repair DNA damages induced by UVC irradiation. This blockage persists as long as the granules are present. A tight correlation between their decay and re-entry into S-phase was observed. However the kinetics of their formation, their low number per cell, their absence of fusion into larger granules, their persistence over 48 hours and their slow decay, all differ from classical SGs induced by arsenite or heat treatment. The induction of these SGs does not correlate with major translation inhibition nor with phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF2α). We propose that a restricted subset of mRNAs coding for proteins implicated in cell cycling are removed from the translational apparatus and are sequestered in a repressed form in SGs.</p></div

UVC-induced granules correspond to SGs.

<p><b>A</b>) Shown are the locations of the SGs protein markers in NIH-3T3 cells grown under normal conditions. <b>B</b>) Cultures were UVC irradiated (10 J/m2) and cells were fixed, permeabilized and processed for immunofluorescence to detect FMRP (red or green), TIA-1/R and TIA-1 (blue), Caprin1 (red), G3BP1 (blue) and PABP1 (red). Note the translocation of G3BP1 and PABP1 into nuclei. Because some of the SGs signals were not easily detected over a high cytoplasmic background, especially in the blue range, shown here are the black and white original images for all single channel pictures, while the merge images are shown in colors. <b>C</b>) Cultures were treated with a buffer containing 1% NP40 to release most of the cytoplasmic fraction and processed for <i>in situ</i> hybridization with poly-dT followed by staining for FMRP using mAb1C3.</p

UVC-induced granules do not correspond to P-bodies.

<p><b>A</b>) Irradiated (10 J/m2) NIH-3T3 cells were fixed, permeabilized and processed for immunofluorescence using antibody against FMRP (green), and the P-bodies markers GW182 (red). <b>B</b>) Double-staining for FMRP (green) and Xrn1 (red). Nuclei were counter-stained with DAPI. <b>C</b>) Number of P-bodies per cell remains constant after UVC irradiation (p not significant), while it almost tripled after arsenite treatment (p<0.0003).</p

Physical comparisons between UVC-induced granules and <i>bone fide</i> SGs.

<p><b>A</b>) Control, UVC-irradiated and arsenite treated NIH-3T3 cells were processed for immunofluorescence to detect FMRP. <b>B</b>) Size of the granules induced by UVC was determined 18 hours post-irradiation (n = 2000) and those induced by arsenite after 60 minutes post-treatment (n = 2000; P value <0.0001). <b>C</b>) Number of granules per cell was determined 18 hours post-irradiation and after 60 minutes arsenite treatment. <b>D</b>) Addition of cycloheximide (50 µg/ml) induced UVC-granules to vanish in less than 60 minutes.</p

UVC-induced SGs are formed independently of eIF2α phosphorylation.

<p><b>A</b>) SGs are present in UVC-irradiated NIH-3T3, MEF^wt and MEF^S51A while they are not formed in MEF^S51A cells treated with arsenite. Cells were fixed, permeabilized, and then processed for immunofluorescence using mAb1C3 against FMRP. <b>B</b>) Immunoblot analyses of MEF^wt showing that eIF2α is not phosphorylated and that HSP90 and 70 are not induced after UVC irradiation, while all three proteins react after arsenite treatment. Tubulin β was used as a control for even loading of samples. C: control; hpt: hours post-treatment.</p

Effects of UVC on translation.

<p><b>A</b>) Polyribosome profiles of control NIH-3T3 and cultures after UVC irradiation with 10 J/m² or exposure to sodium arsenite. Cells were extracted 18h after UV irradiation or after 30 minutes of arsenite treatment and the cytoplasmic fractions analyzed by sedimentation in sucrose density gradients. The sedimentation position of the 80S ribosomes is indicated by an arrow. <b>B</b>) Newly synthesized proteins detected by incorporation of ³⁵S-methionin/cystein in cells exposed to sodium arsenite or irradiated with UVC. Cells were labelled 30 minutes in the presence of 50 µCi/ml [³⁵S]-methionine/cystein in methionine-free DMEM. Total cell extracts (∼40 µg protein) were analyzed on a 11% SDS-PAGE. Upper panel: Coomassie blue staining. Lower panel: the same gel was dried and exposed to an X-ray film. Stars indicate the positions of hsp90 and hsp70.</p

UVC induces the formation of small cytoplasmic granules and affect cell proliferation.

<p><b>A</b>) NIH-3T3 cells were treated with 5, 10, 20, and 30 J/m² UV at 254 nm. Eighteen hours post-irradiation, cells were fixed, permeabilized, and processed for immunofluorescence using mAb1C3 against FMRP. Nuclei were counterstained with DAPI. The percentage of cells containing granules was determined from four different experiments each at different irradiation doses. A total of 2000 cells was observed for each dose. Error represents 1 SD calculated from 4 sets of independent experiments. <b>B</b>) Proliferation kinetics of NIH-3T3 seeded on coverslips with engraved grids after irradiation at different doses of UVC (5, 10, 20 and 30 J/m²). Cells were photographed with a camera on an inverted microscope equipped with phase contrast. The number of cells on each grid was counted from five different experiments. Error represents 1 SD calculated from 5 sets of independent experiments. Stars indicate 100% confluence. <b>C</b>) Cell cycle distribution of NIH-3T3 showing a complete G₁ arrest in cells exposed to 10 J/m² and fragmented DNA (apoptotic: Apo) after a 20 J/m² irradiation. Cells were analyzed 18 h after irradiation. <b>D</b>) Correlation between the presence of granules and DNA synthesis in cells exposed to 10 J/m². Quiescent serum-starved NIH-3T3 cells were irradiated and then serum stimulated. At different times after irradiation, cells were fixed, permeabilized, and then processed for immunofluorescence using antibodies against FMRP. The number of cells containing granules was counted from three different experiments containing a total of 3000 cells. Also shown is the rate of incorporation of [³H]-Thd into trichloroacetic acid precipitable material after a 30 minutes pulse labeling. Insert i): time course of serum-induced DNA synthesis in synchronized control cells. Insert ii): FACS analysis showing cells re-entering S-phase at 48 hours post-irradiation. <b>E</b>) Eighteen hours post-irradiation, NIH-3T3 cells were fixed, permeabilized, and processed for immunofluorescence using antibodies against FMRP (red) and γ-H2AX (green) to determine the formation of FMRP granules and the extend of double-strand breaks induced by UVC irradiation. Nuclei were counterstained with DAPI. The arrowhead in the merged image (right panel) points to a cell that shows very weak γ-H2AX staining and no FMRP granules.</p

Effects of Leptomycin B on nuclear FMRP localization.

<p>Hela cells were maintained in normal conditions (A) or treated with 2 ng/ml LMB for 18 h (B), and then processed for immunofluorescence to localize FMRP (red) and Coilin (green). Nuclei were counterstained with DAPI.</p

Effects of Leptomycin B on cytoplasmic and nuclear GFP-FMRP localizations.

<p>Control and transfected HeLa cells with vectors coding for GFP-ISO7 (A) and GFP-ISO6 (B) were maintained in normal conditions or treated with 2 ng/ml LMB for 20 h, and then processed for immunofluorescence to localize FMRP (green) and Coilin (red). Nuclei were stained with DAPI.</p

FMRP is present in the isolated nuclear fraction but not in nuclei.

<p>(A) Total, cytoplasmic, and nuclear cytoplasmic fractions from HeLa cells were loaded in equal ratios as well as one overloaded nuclear fraction and analyzed by immunoblotting with mAb1C3 to determine the distribution of FMRP. Nuc+ refers to concentrated (20 µg) nuclear protein. (B) Double immunofluorescent localization of FMRP with IgYC10 (red) and Coilin (green) after gentle lysis of the cells <i>in situ</i>. Nuclei were counterstained with DAPI. (C) Double immunofluorescent staining of FMRP with IgYC10 (red) and cold-resistant microtubule network revealed with an anti-tubulin antibody (green). Nuclei were counterstained with DAPI. Due to the three dimensional distribution of microtubules, images were taken by conventional epifluorescent microscopy to reveal the microtubule framework.</p