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Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.

Rothé B, Leal-Esteban L, Bernet F, Urfer S, Doerr N, Weimbs T, Iwaszkiewicz J, Constam DB - Mol. Cell. Biol. (2015)

Bottom Line: In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway.Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects.We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.

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Related in: MedlinePlus

SAM polymerization is required for Bicc1 accumulation and silencing activity. (A) Time course analysis of HA-Bicc1 WT and mutants expressed in HEK293T cells after CHX treatment. HA-Bicc1 protein levels were compared with γ-tubulin levels by Western blotting at 0, 8, 24, 32, 48, and 56 h. The relative level of each protein is presented in the graph at the bottom, and the estimated half-life is given on the right. AU, arbitrary units. (B) Level of the HA-Bicc1 WT and mutants upon transfection with a single dose (×1) or a double dose (×2) of DNA encoding HA-Bicc1. A double dose of transfected DNA encoding HA-Bicc1 mutD, the ΔSAM mutant, or the bpk mutant is required to obtain a protein level comparable to that of the HA-Bicc1 WT. The relative percentage of WT Bicc1 is indicated for each condition. γ-Tubulin was used for normalization. (C and D) Silencing of AC6 and PKIα 3′ UTR luciferase reporters by WT or polymerization mutant Bicc1 in HEK293T cells. β-Galactosidase was used as a control for normalization. Error bars show SEMs. *, P < 0.005. (E) Induction of the TOPflash reporter of Wnt signaling by Dishevelled 2 (Dvl2) in transfected HEK293T cells is inhibited by both WT and polymerization mutant Bicc1. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
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Figure 6: SAM polymerization is required for Bicc1 accumulation and silencing activity. (A) Time course analysis of HA-Bicc1 WT and mutants expressed in HEK293T cells after CHX treatment. HA-Bicc1 protein levels were compared with γ-tubulin levels by Western blotting at 0, 8, 24, 32, 48, and 56 h. The relative level of each protein is presented in the graph at the bottom, and the estimated half-life is given on the right. AU, arbitrary units. (B) Level of the HA-Bicc1 WT and mutants upon transfection with a single dose (×1) or a double dose (×2) of DNA encoding HA-Bicc1. A double dose of transfected DNA encoding HA-Bicc1 mutD, the ΔSAM mutant, or the bpk mutant is required to obtain a protein level comparable to that of the HA-Bicc1 WT. The relative percentage of WT Bicc1 is indicated for each condition. γ-Tubulin was used for normalization. (C and D) Silencing of AC6 and PKIα 3′ UTR luciferase reporters by WT or polymerization mutant Bicc1 in HEK293T cells. β-Galactosidase was used as a control for normalization. Error bars show SEMs. *, P < 0.005. (E) Induction of the TOPflash reporter of Wnt signaling by Dishevelled 2 (Dvl2) in transfected HEK293T cells is inhibited by both WT and polymerization mutant Bicc1. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.

Mentions: Since the Bicc1 mutant D apparently did not freely accumulate as a monomer in density fractionation gradients, we asked whether polymerization influences the protein half-life. Translation inhibition by cycloheximide (CHX) followed by a time course analysis by immunoblotting showed that wild-type HA-Bicc1 remained stable over the entire chase period (56 h). In contrast, the half-lives of the HA-Bicc1 mutD and Bicc1ΔSAM proteins were reduced to 34 and 29 h, respectively (Fig. 6A), suggesting that SAM polymerization increases Bicc1 stability. Therefore, in all subsequent assays evaluating their functions, we doubled the dose of transfected DNA for Bicc1 mutants in order to reach expression levels comparable to those of wild-type HA-Bicc1 (Fig. 6B). In particular, to determine whether polymerization is necessary for mRNA silencing, we compared the potential of wild-type HA-Bicc1 and mutant D to silence the 3′ UTR sequences of AC6 and PKIα mRNAs in luciferase reporter assays. HA-Bicc1 significantly repressed the expression of these luciferase reporters, as described previously (27). In contrast, both HA-Bicc1ΔSAM and HA-Bicc1 mutD failed to repress these targets even at an elevated dosage (Fig. 6C and D), indicating that the silencing activity of Bicc1 directly depends on SAM polymerization.


Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.

Rothé B, Leal-Esteban L, Bernet F, Urfer S, Doerr N, Weimbs T, Iwaszkiewicz J, Constam DB - Mol. Cell. Biol. (2015)

SAM polymerization is required for Bicc1 accumulation and silencing activity. (A) Time course analysis of HA-Bicc1 WT and mutants expressed in HEK293T cells after CHX treatment. HA-Bicc1 protein levels were compared with γ-tubulin levels by Western blotting at 0, 8, 24, 32, 48, and 56 h. The relative level of each protein is presented in the graph at the bottom, and the estimated half-life is given on the right. AU, arbitrary units. (B) Level of the HA-Bicc1 WT and mutants upon transfection with a single dose (×1) or a double dose (×2) of DNA encoding HA-Bicc1. A double dose of transfected DNA encoding HA-Bicc1 mutD, the ΔSAM mutant, or the bpk mutant is required to obtain a protein level comparable to that of the HA-Bicc1 WT. The relative percentage of WT Bicc1 is indicated for each condition. γ-Tubulin was used for normalization. (C and D) Silencing of AC6 and PKIα 3′ UTR luciferase reporters by WT or polymerization mutant Bicc1 in HEK293T cells. β-Galactosidase was used as a control for normalization. Error bars show SEMs. *, P < 0.005. (E) Induction of the TOPflash reporter of Wnt signaling by Dishevelled 2 (Dvl2) in transfected HEK293T cells is inhibited by both WT and polymerization mutant Bicc1. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
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Figure 6: SAM polymerization is required for Bicc1 accumulation and silencing activity. (A) Time course analysis of HA-Bicc1 WT and mutants expressed in HEK293T cells after CHX treatment. HA-Bicc1 protein levels were compared with γ-tubulin levels by Western blotting at 0, 8, 24, 32, 48, and 56 h. The relative level of each protein is presented in the graph at the bottom, and the estimated half-life is given on the right. AU, arbitrary units. (B) Level of the HA-Bicc1 WT and mutants upon transfection with a single dose (×1) or a double dose (×2) of DNA encoding HA-Bicc1. A double dose of transfected DNA encoding HA-Bicc1 mutD, the ΔSAM mutant, or the bpk mutant is required to obtain a protein level comparable to that of the HA-Bicc1 WT. The relative percentage of WT Bicc1 is indicated for each condition. γ-Tubulin was used for normalization. (C and D) Silencing of AC6 and PKIα 3′ UTR luciferase reporters by WT or polymerization mutant Bicc1 in HEK293T cells. β-Galactosidase was used as a control for normalization. Error bars show SEMs. *, P < 0.005. (E) Induction of the TOPflash reporter of Wnt signaling by Dishevelled 2 (Dvl2) in transfected HEK293T cells is inhibited by both WT and polymerization mutant Bicc1. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
Mentions: Since the Bicc1 mutant D apparently did not freely accumulate as a monomer in density fractionation gradients, we asked whether polymerization influences the protein half-life. Translation inhibition by cycloheximide (CHX) followed by a time course analysis by immunoblotting showed that wild-type HA-Bicc1 remained stable over the entire chase period (56 h). In contrast, the half-lives of the HA-Bicc1 mutD and Bicc1ΔSAM proteins were reduced to 34 and 29 h, respectively (Fig. 6A), suggesting that SAM polymerization increases Bicc1 stability. Therefore, in all subsequent assays evaluating their functions, we doubled the dose of transfected DNA for Bicc1 mutants in order to reach expression levels comparable to those of wild-type HA-Bicc1 (Fig. 6B). In particular, to determine whether polymerization is necessary for mRNA silencing, we compared the potential of wild-type HA-Bicc1 and mutant D to silence the 3′ UTR sequences of AC6 and PKIα mRNAs in luciferase reporter assays. HA-Bicc1 significantly repressed the expression of these luciferase reporters, as described previously (27). In contrast, both HA-Bicc1ΔSAM and HA-Bicc1 mutD failed to repress these targets even at an elevated dosage (Fig. 6C and D), indicating that the silencing activity of Bicc1 directly depends on SAM polymerization.

Bottom Line: In addition, defective polymerization decreases Bicc1 stability and thus indirectly attenuates inhibition of Dishevelled 2 in the Wnt/β-catenin pathway.Importantly, aberrant C-terminal extension of the SAM domain in bpk mutant Bicc1 phenocopied these defects.We conclude that polymerization is a novel disease-relevant mechanism both to stabilize Bicc1 and to present associated mRNAs in specific silencing platforms.

View Article: PubMed Central - PubMed

Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.

Show MeSH
Related in: MedlinePlus