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Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ.

Bersani C, Xu LD, Vilborg A, Lui WO, Wiman KG - Oncogene (2014)

Bottom Line: We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells.We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53.We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT
Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

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FAS mRNA colocalizes with Wig-1 protein in SGs. RNA FISH staining for FAS mRNA together with anti-Wig-1 (a), anti-TIAR (b) or anti-HEDLS (c) antibody in untreated or treated (0.5 mM arsenite) U2OS cells. TIAR and HEDLS proteins were used as SG and processing body marker, respectively. Arrows indicate the positions of overlapping FAS mRNA and Wig-1 or TIAR proteins. Scale bars, 20 μm. Blue dye shows DAPI DNA staining. Representative images from one of two independent experiments are shown. (d) Model for the role of Wig-1 in regulation of cell death and survival in the p53 stress response. (e) Model for Wig-1-mediated post-transcriptional regulation of FAS mRNA.
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fig6: FAS mRNA colocalizes with Wig-1 protein in SGs. RNA FISH staining for FAS mRNA together with anti-Wig-1 (a), anti-TIAR (b) or anti-HEDLS (c) antibody in untreated or treated (0.5 mM arsenite) U2OS cells. TIAR and HEDLS proteins were used as SG and processing body marker, respectively. Arrows indicate the positions of overlapping FAS mRNA and Wig-1 or TIAR proteins. Scale bars, 20 μm. Blue dye shows DAPI DNA staining. Representative images from one of two independent experiments are shown. (d) Model for the role of Wig-1 in regulation of cell death and survival in the p53 stress response. (e) Model for Wig-1-mediated post-transcriptional regulation of FAS mRNA.

Mentions: The CCR4–NOT complex is known to regulate gene expression in response to stress.26 To explore whether CNOT6 interacts with Wig-1 upon stress, we determined whether CNOT6 colocalizes with Wig-1 after arsenite-induced stress using coimmunostaining in HCT116 p53+/+ cells. Under unstressed conditions, both proteins localize uniformly throughout the cytoplasm, although Wig-1 is predominantly nuclear. As the uniform cytoplasmic distribution makes colocalization studies difficult, we used arsenite, an inducer of stress granules (SGs) and processing bodies, for these experiments. The formation of these structures facilitates protein colocalization studies. We found that arsenite caused relocalization of both Wig-1 and CNOT6 to SGs, as shown by coimmunostaining with the SG marker TIAR (Figures 5c and d) but not with the processing body marker HEDLS (Supplementary Figure 5a). Arsenite-induced stress did not affect Wig-1 or p53 protein levels (Supplementary Figure 6), suggesting that the observed Wig-1 accumulation in SGs is indeed the result of cytoplasmic re-distribution. To investigate whether FAS mRNA also colocalizes with Wig-1 to SGs, U2OS cells were treated with arsenite and FAS mRNA was detected by RNA-FISH using an FAS mRNA fluorescent probe. Coimmunostaining with antibodies against either endogenous Wig-1 (Figure 6a) or TIAR (Figure 6b) confirmed colocalization of FAS mRNA with Wig-1 in SGs. No FAS mRNA colocalized with HEDLS in the processing body.(Figure 6c; see Supplementary Figure 5b–d for additional examples and negative control data with the sense FAS probe). These results suggest that Wig-1 and CNOT6 proteins interact with FAS mRNA in cytoplasmic SGs after cellular stress.


Wig-1 regulates cell cycle arrest and cell death through the p53 targets FAS and 14-3-3σ.

Bersani C, Xu LD, Vilborg A, Lui WO, Wiman KG - Oncogene (2014)

FAS mRNA colocalizes with Wig-1 protein in SGs. RNA FISH staining for FAS mRNA together with anti-Wig-1 (a), anti-TIAR (b) or anti-HEDLS (c) antibody in untreated or treated (0.5 mM arsenite) U2OS cells. TIAR and HEDLS proteins were used as SG and processing body marker, respectively. Arrows indicate the positions of overlapping FAS mRNA and Wig-1 or TIAR proteins. Scale bars, 20 μm. Blue dye shows DAPI DNA staining. Representative images from one of two independent experiments are shown. (d) Model for the role of Wig-1 in regulation of cell death and survival in the p53 stress response. (e) Model for Wig-1-mediated post-transcriptional regulation of FAS mRNA.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: FAS mRNA colocalizes with Wig-1 protein in SGs. RNA FISH staining for FAS mRNA together with anti-Wig-1 (a), anti-TIAR (b) or anti-HEDLS (c) antibody in untreated or treated (0.5 mM arsenite) U2OS cells. TIAR and HEDLS proteins were used as SG and processing body marker, respectively. Arrows indicate the positions of overlapping FAS mRNA and Wig-1 or TIAR proteins. Scale bars, 20 μm. Blue dye shows DAPI DNA staining. Representative images from one of two independent experiments are shown. (d) Model for the role of Wig-1 in regulation of cell death and survival in the p53 stress response. (e) Model for Wig-1-mediated post-transcriptional regulation of FAS mRNA.
Mentions: The CCR4–NOT complex is known to regulate gene expression in response to stress.26 To explore whether CNOT6 interacts with Wig-1 upon stress, we determined whether CNOT6 colocalizes with Wig-1 after arsenite-induced stress using coimmunostaining in HCT116 p53+/+ cells. Under unstressed conditions, both proteins localize uniformly throughout the cytoplasm, although Wig-1 is predominantly nuclear. As the uniform cytoplasmic distribution makes colocalization studies difficult, we used arsenite, an inducer of stress granules (SGs) and processing bodies, for these experiments. The formation of these structures facilitates protein colocalization studies. We found that arsenite caused relocalization of both Wig-1 and CNOT6 to SGs, as shown by coimmunostaining with the SG marker TIAR (Figures 5c and d) but not with the processing body marker HEDLS (Supplementary Figure 5a). Arsenite-induced stress did not affect Wig-1 or p53 protein levels (Supplementary Figure 6), suggesting that the observed Wig-1 accumulation in SGs is indeed the result of cytoplasmic re-distribution. To investigate whether FAS mRNA also colocalizes with Wig-1 to SGs, U2OS cells were treated with arsenite and FAS mRNA was detected by RNA-FISH using an FAS mRNA fluorescent probe. Coimmunostaining with antibodies against either endogenous Wig-1 (Figure 6a) or TIAR (Figure 6b) confirmed colocalization of FAS mRNA with Wig-1 in SGs. No FAS mRNA colocalized with HEDLS in the processing body.(Figure 6c; see Supplementary Figure 5b–d for additional examples and negative control data with the sense FAS probe). These results suggest that Wig-1 and CNOT6 proteins interact with FAS mRNA in cytoplasmic SGs after cellular stress.

Bottom Line: We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells.We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53.We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR.

View Article: PubMed Central - PubMed

Affiliation: Cancer Center Karolinska (CCK), Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden.

ABSTRACT
Wig-1, also known as ZMAT3, is a p53 target gene that encodes an RNA-binding zinc-finger protein involved in the regulation of mRNA stability through binding to AU-rich elements (AREs). We have used microarray analysis to identify novel Wig-1 target mRNAs. We identified 2447 transcripts with >fourfold differential expression between Wig-1 and control small interfering (si)RNA-treated HCT116 cells. Several p53 target genes were among the deregulated transcripts. We found that Wig-1 regulates FAS and 14-3-3σ mRNA independently of p53. We show that Wig-1 binds to FAS mRNA 3'-UTR and decreases its stability through an ARE in the 3'-UTR. Depletion of Wig-1 was associated with increased cell death and reduced cell cycle arrest upon DNA damage. Our results suggest a role of Wig-1 as a survival factor that directs the p53 stress response toward cell cycle arrest rather than apoptosis through the regulation of FAS and 14-3-3σ mRNA levels.

Show MeSH
Related in: MedlinePlus