Limits...
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.

Show MeSH

Related in: MedlinePlus

Wig-1 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) (a). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control (b). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes (c) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. (d). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in (c) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion (e). Columns and error bars in (a), (b) and (e) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150987&req=5

fig4: Wig-1 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) (a). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control (b). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes (c) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. (d). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in (c) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion (e). Columns and error bars in (a), (b) and (e) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.

Mentions: To examine binding of Wig-1 to FAS mRNA, we performed RNA-immunoprecipitation assays in HCT116 and Saos-2 cells expressing Flag-tagged Wig-1, as well as Saos-2 cells expressing a mutant Wig-1 protein that lacks the first zinc-finger (Wig-1ZF1pm) and is therefore unable to bind to RNA.13 Quantification of coimmunoprecipitated FAS mRNA revealed a four- to fivefold enrichment in immunoprecipitates of Flag-tagged wild-type Wig-1 as compared with empty vector control (Figure 4a). There was no significant enrichment of FAS mRNA in Flag-Wig-1ZF1pm immunoprecipitates (Figure 4a), indicating that zinc-finger 1 is required for Wig-1 binding to FAS mRNA.


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 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) (a). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control (b). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes (c) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. (d). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in (c) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion (e). Columns and error bars in (a), (b) and (e) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig4: Wig-1 binds to the 3′-UTR of FAS mRNA and regulates its stability through the ARE. RNA immunoprecipitation was performed in HCT116 cells transiently transfected with pCMVtag2b (Flag) or pCMVtag2bhWig-1 (Flag-Wig-1) and in Saos-2 TetON cells without insert (Ctrl) or Saos-2 TetON cells stably transfected with either Flag-tagged wt Wig-1 (Wig-1) or a Flag-tagged Wig-1 zinc-finger 1 point mutant that cannot bind to RNA (Wig-1ZF1pm) (a). Wig-1 was precipitated with anti-Flag beads, and bound RNA was purified and quantified by qRT–PCR. GAPDH mRNA levels were used as internal control. Labeling of nascent RNA with 4-sU, RNA extraction, conjugation of the 4-sU to biotin and separation of nascent and older RNA with streptavidin beads followed by qRT–PCR analysis of the two populations separately showed no significant variation in the 4-sU-labeled RNA, whereas we observed an increase in the older, unlabeled RNA population after Wig-1 knockdown. GAPDH was used as internal control (b). Biotin pull-down assay using FAS 5′-UTR-ORF, FAS 3′-UTR, FAS 3′-UTR distal and FAS 3′-UTR distal dARE probes (c) followed by western blotting for Wig-1 shows that Wig-1 binds to the ARE in the 3′-UTR of FAS. (d). Representative image from one of two independent experiments. To determine whether the ARE is required for Wig-1 regulation of FAS mRNA, we generated the constructs 2–5 described in (c) or the p53 3′-UTR lacking the ARE as negative control and cloned them downstream of the Renilla reporter into the psiCheck-2 vector. Luciferase assays confirmed that the ARE in the 3′-UTR of FAS mRNA is essential for regulation by Wig-1, as constructs lacking this ARE do not show increased activity after Wig-1 depletion (e). Columns and error bars in (a), (b) and (e) represent the mean±s.d.; n=3; ***P<0.001; **P<0.01; *P<0.05.
Mentions: To examine binding of Wig-1 to FAS mRNA, we performed RNA-immunoprecipitation assays in HCT116 and Saos-2 cells expressing Flag-tagged Wig-1, as well as Saos-2 cells expressing a mutant Wig-1 protein that lacks the first zinc-finger (Wig-1ZF1pm) and is therefore unable to bind to RNA.13 Quantification of coimmunoprecipitated FAS mRNA revealed a four- to fivefold enrichment in immunoprecipitates of Flag-tagged wild-type Wig-1 as compared with empty vector control (Figure 4a). There was no significant enrichment of FAS mRNA in Flag-Wig-1ZF1pm immunoprecipitates (Figure 4a), indicating that zinc-finger 1 is required for Wig-1 binding to FAS mRNA.

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