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. 2010 May;38(9):3005-18.
doi: 10.1093/nar/gkq004. Epub 2010 Jan 27.

Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin

Roberta Busà  1 , Raffaele Geremia, Claudio Sette
Affiliations

Genotoxic stress causes the accumulation of the splicing regulator Sam68 in nuclear foci of transcriptionally active chromatin

Roberta Busà et al. Nucleic Acids Res. 2010 May.

Abstract

DNA-damaging agents cause a multifaceted cellular stress response. Cells set in motion either repair mechanisms or programmed cell death pathways, depending on the extent of the damage and on their ability to withstand it. The RNA-binding protein (RBP) Sam68, which is up-regulated in prostate carcinoma, promotes prostate cancer cell survival to genotoxic stress. Herein, we have investigated the function of Sam68 in this cellular response. Mitoxantrone (MTX), a topoisomerase II inhibitor, induced relocalization of Sam68 from the nucleoplasm to nuclear granules, together with several other RBPs involved in alternative splicing, such as TIA-1, hnRNP A1 and the SR proteins SC35 and ASF/SF2. Sam68 accumulation in nuclear stress granules was independent of signal transduction pathways activated by DNA damage. Using BrU labelling and immunofluorescence, we demonstrate that MTX-induced nuclear stress granules are transcriptionally active foci where Sam68 and the phosphorylated form of RNA polymerase II accumulate. Finally, we show that MTX-induced relocalization of Sam68 correlates with changes in alternative splicing of its mRNA target CD44, and that MTX-induced CD44 splicing depends on Sam68 expression. These results strongly suggest that Sam68 is part of a RNA-mediated stress response of the cell that modulates alternative splicing in response to DNA damage.

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Figures

Figure 1.
Figure 1.
Sam68 relocalizes in subcellular compartments after genotoxic stress. (A) Time course treatment of PC3 cells with 5 µM MTX. Treated cells were stained with an anti-Sam68-specific antibody and analysed by immunofluorescence by confocal microscopy. The localization of Sam68 around the nucleoli (2 h MTX) and in large nuclear granules (8–24 h MTX) is indicated by white arrows. Scale bar = 15 µm. (B) Graph of the percentage of PC3 cells treated showing ring-shaped structures and granules after MTX treatment (5 µM, 2–24 h). (C) Phase contrast images of control and MTX-treated cells (5 µM). (D) Cell extracts (10 µg) from PC3 treated as in panel A were analysed by SDS–PAGE and western blot with the indicated antibodies. (E) PC3 cells were treated for 16 h with 80 µM cisplatin, fixed and stained with anti-Sam68 antibody. The white arrow indicates a Sam68 granule in treated cells. Scale bar = 15 µm. (F) Confocal images of PC3 cells after prolonged treatment (24 h) with 5 µM MTX indicating the translocation of Sam68 in the cytoplasm. Scale bar = 15 µm.
Figure 2.
Figure 2.
Sam68 colocalizes with stress-response RNA-binding proteins after genotoxic stress. PC3 cells were treated with 5 µM MTX (2–24 h) and co-stained with Sam68 and hnRNP A1 (A) or TIA-1 (B) specific antibodies and analysed by confocal microscopy as in Figure 1. The merge panels show colocalization of Sam68 and hnRNP A1 or TIA-1 to the same nuclear granules. Scale bar = 10 µm.
Figure 3.
Figure 3.
SR proteins colocalize with Sam68 in nuclear granules after genotoxic stress. Confocal analyses of PC3 cells treated with 5 µM MTX (2–24 h) and co-stained with Sam68 and SC35 (A) or ASF/SF2 (B). Sam68 colocalized to the same stress-induced nuclear granules as SC35 and ASF/SF2 after 8–24 h of MTX treatment. Stress-induced nuclear granules are pointed by white arrows. Scale bar = 10 µm.
Figure 4.
Figure 4.
The Sam68 GSG homodimerization and RNA-binding domain is sufficient for relocalization after genotoxic stress. PC3 cells were transiently transfected with full-length GFP-Sam68 (A) or with GFP-Sam68GSG (B) and treated with 5 µM MTX. After 24 h, the cells were fixed and analysed by confocal microscope. Scale bar = 10 µm.
Figure 5.
Figure 5.
Genotoxic stress induced by MTX causes an enrichment of the splicing factors Sam68 and hnRNP A1 in the transcriptionally active fractions. (A) Western blot analysis of chromatin fractions obtained from PC3 cells treated with 0.5 or 5 µM MTX. Distribution of proteins in total digested nuclei (T) and in chromatin fractions (supernatants S1–S2 and pellet, P) was analysed with the indicated antibodies. Lamin-B was used as nuclear matrix marker. Staining for histone H3 was performed as loading control. (B and C) Densitometric analysis of the distribution of Sam68 (B) and TFIIH p89 (C) in S1–S2–P chromatin fractions from three experiments is expressed as percentage of the levels of these proteins in the total digested nuclei (T) (mean ± SD; *P < 0.05; **P < 0.01).
Figure 6.
Figure 6.
RNAPII and Sam68 colocalize in genotoxic stress-induced nuclear granules. (A) PC3 cells untreated (control) or treated with 5 µM MTX (2–24 h), were stained with a RNAPII antibody (H5) specific for the phosphorylated serine2 and analysed by confocal microscopy. (B) Co-staining of Sam68 and phosphorylated RNAPII (H5) in PC3 cells treated as in panel A. Scale bar = 10 µm.
Figure 7.
Figure 7.
Sam68 nuclear granules induced by genotoxic stress are transcriptionally active foci. (A) BrU-incorporation assay in PC3 cells untreated (control) or treated with 5 µM MTX (2–24 h). PC3 cells were stained with anti-Sam68 and BrU specific antibodies. Confocal analysis revealed that Sam68 MTX-induced nuclear granules incorporate BrU. (B). PC3 cells were treated for 2 h with 5 µM MTX, and allowed to recover 24 h after damage. At the end of incubation, cells were fixed and stained with anti-Sam68 and BrU specific antibodies. Scale bar = 10 µm.
Figure 8.
Figure 8.
Genotoxic stress induced by MTX affects CD44 alternative splicing in a Sam68-dependent manner. (A) control pLKO and pLKO-si-Sam68 PC3 cells were treated for 2 h with 0.5 (white columns) or 5 µM MTX (dim grey columns), allowed to recover from stress for 24 h and analysed for the levels of endogenous CD44 v5 exon inclusion by real-time PCR. CD44 v5 levels in untreated cells (dark grey columns) were set as 1 and MTX-induced v5 inclusion was calculated as fold increase. Samples were normalized for CD44 constitutive isoforms levels (see details under ‘Materials and Methods’ section). Data are expressed as mean-fold induction ±SD from three separate experiments. (B) Western blot analysis of cell extracts (15 µg) from the PC3 clones confirmed down-regulation of Sam68 levels in pLKO-si-Sam68 cells. (C) pLKO and pLKO-si-Sam68 PC3 cells were treated with 5 µM MTX for 2 h, allowed to recover and transiently transfected with pETv5Luc minigene in the presence (+) or absence (−) of murine mycSam68. The v5-Luc activity in untreated cells was set as 100% (dark grey columns) and v5-Luc activity in MTX-treated cells without (dim grey columns) or with (white column) murine mycSam68 was expressed as percentage respect to untreated cells. Data are expressed as mean-fold induction ±SD from three separate experiments. (D) Western blot analysis of the pLKO clones cell extracts 15 µg) confirmed down-regulation of Sam68 levels in pLKO-si-Sam68 cells and the overexpression of mycSam68. Protein loading was normalized on hnRNP A1 levels both in panel B an D.
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