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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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Related in: MedlinePlus

Wig-1 interacts with deadenylase subunit CNOT6. Knockdown of the key components of the ARE-mediated mRNA decay pathway CNOT6, DCP1a, PM/Scl-75 and XRN1 was performed in HCT116 p53−/− cells, and the levels of FAS mRNA were assessed through qRT–PCR and compared with levels upon Wig-1 depletion. The figure shows that FAS mRNA levels are increased in a comparable manner following knockdown of CNOT6 or Wig-1 (no significant difference), suggesting that the Wig-1 effect on FAS mRNA is exerted at the level of poly(A) tail deadenylation (a). Protein lysates from HCT116 p53+/+ or HCT116 p53−/− cells transfected with pCMVtag2b or pCMVtag2bhWig-1 were immunoprecipitated with a Flag antibody or CNOT6 antibody cross-linked to Dynabeads Protein G, followed by western blotting with indicated antibodies (b). Coimmunostaining of endogenous Wig-1 (green) and CNOT6 (red) (c) and Wig-1 (green) and SGs marker TIAR (red) (d) in HCT116 p53+/+ cells after 30 min of treatment with 0.5 mM arsenite shows colocalization in cytoplasmic SGs. Arrows indicate the positions of overlapping Wig-1 and CNOT6 foci or Wig-1 and TIAR. Scale bars, 20 μm. DAPI shows DNA staining.
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fig5: Wig-1 interacts with deadenylase subunit CNOT6. Knockdown of the key components of the ARE-mediated mRNA decay pathway CNOT6, DCP1a, PM/Scl-75 and XRN1 was performed in HCT116 p53−/− cells, and the levels of FAS mRNA were assessed through qRT–PCR and compared with levels upon Wig-1 depletion. The figure shows that FAS mRNA levels are increased in a comparable manner following knockdown of CNOT6 or Wig-1 (no significant difference), suggesting that the Wig-1 effect on FAS mRNA is exerted at the level of poly(A) tail deadenylation (a). Protein lysates from HCT116 p53+/+ or HCT116 p53−/− cells transfected with pCMVtag2b or pCMVtag2bhWig-1 were immunoprecipitated with a Flag antibody or CNOT6 antibody cross-linked to Dynabeads Protein G, followed by western blotting with indicated antibodies (b). Coimmunostaining of endogenous Wig-1 (green) and CNOT6 (red) (c) and Wig-1 (green) and SGs marker TIAR (red) (d) in HCT116 p53+/+ cells after 30 min of treatment with 0.5 mM arsenite shows colocalization in cytoplasmic SGs. Arrows indicate the positions of overlapping Wig-1 and CNOT6 foci or Wig-1 and TIAR. Scale bars, 20 μm. DAPI shows DNA staining.

Mentions: To delineate the mechanism of Wig-1-dependent regulation of FAS mRNA stability, we silenced known components of the ARE-mediated mRNA decay pathway. We knocked down the expression of CNOT6 (subunit of the CCR4–NOT deadenylation complex), PmScl-75 (exonuclease of the exosome), DCP1a (5′-decapping enzyme) and XRN1 (5′- to 3′- exoribonuclease). As shown in Figure 5a, Wig-1 or CNOT6 knockdown in HCT116 p53−/− cells led to a similar increase in FAS mRNA levels, whereas the increase of FAS mRNA after silencing of other degradation pathway enzymes was significantly lower. This result suggests that CNOT6 is responsible for degrading FAS mRNA. We propose that Wig-1 recruits the CCR4–NOT complex to FAS mRNA and thereby triggers its deadenylation, leading to inhibition of FAS expression.


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)

Wig-1 interacts with deadenylase subunit CNOT6. Knockdown of the key components of the ARE-mediated mRNA decay pathway CNOT6, DCP1a, PM/Scl-75 and XRN1 was performed in HCT116 p53−/− cells, and the levels of FAS mRNA were assessed through qRT–PCR and compared with levels upon Wig-1 depletion. The figure shows that FAS mRNA levels are increased in a comparable manner following knockdown of CNOT6 or Wig-1 (no significant difference), suggesting that the Wig-1 effect on FAS mRNA is exerted at the level of poly(A) tail deadenylation (a). Protein lysates from HCT116 p53+/+ or HCT116 p53−/− cells transfected with pCMVtag2b or pCMVtag2bhWig-1 were immunoprecipitated with a Flag antibody or CNOT6 antibody cross-linked to Dynabeads Protein G, followed by western blotting with indicated antibodies (b). Coimmunostaining of endogenous Wig-1 (green) and CNOT6 (red) (c) and Wig-1 (green) and SGs marker TIAR (red) (d) in HCT116 p53+/+ cells after 30 min of treatment with 0.5 mM arsenite shows colocalization in cytoplasmic SGs. Arrows indicate the positions of overlapping Wig-1 and CNOT6 foci or Wig-1 and TIAR. Scale bars, 20 μm. DAPI shows DNA staining.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150987&req=5

fig5: Wig-1 interacts with deadenylase subunit CNOT6. Knockdown of the key components of the ARE-mediated mRNA decay pathway CNOT6, DCP1a, PM/Scl-75 and XRN1 was performed in HCT116 p53−/− cells, and the levels of FAS mRNA were assessed through qRT–PCR and compared with levels upon Wig-1 depletion. The figure shows that FAS mRNA levels are increased in a comparable manner following knockdown of CNOT6 or Wig-1 (no significant difference), suggesting that the Wig-1 effect on FAS mRNA is exerted at the level of poly(A) tail deadenylation (a). Protein lysates from HCT116 p53+/+ or HCT116 p53−/− cells transfected with pCMVtag2b or pCMVtag2bhWig-1 were immunoprecipitated with a Flag antibody or CNOT6 antibody cross-linked to Dynabeads Protein G, followed by western blotting with indicated antibodies (b). Coimmunostaining of endogenous Wig-1 (green) and CNOT6 (red) (c) and Wig-1 (green) and SGs marker TIAR (red) (d) in HCT116 p53+/+ cells after 30 min of treatment with 0.5 mM arsenite shows colocalization in cytoplasmic SGs. Arrows indicate the positions of overlapping Wig-1 and CNOT6 foci or Wig-1 and TIAR. Scale bars, 20 μm. DAPI shows DNA staining.
Mentions: To delineate the mechanism of Wig-1-dependent regulation of FAS mRNA stability, we silenced known components of the ARE-mediated mRNA decay pathway. We knocked down the expression of CNOT6 (subunit of the CCR4–NOT deadenylation complex), PmScl-75 (exonuclease of the exosome), DCP1a (5′-decapping enzyme) and XRN1 (5′- to 3′- exoribonuclease). As shown in Figure 5a, Wig-1 or CNOT6 knockdown in HCT116 p53−/− cells led to a similar increase in FAS mRNA levels, whereas the increase of FAS mRNA after silencing of other degradation pathway enzymes was significantly lower. This result suggests that CNOT6 is responsible for degrading FAS mRNA. We propose that Wig-1 recruits the CCR4–NOT complex to FAS mRNA and thereby triggers its deadenylation, leading to inhibition of FAS expression.

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