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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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SAM polymerization is required for Bicc1 clustering. (A) Density fractionation of WT and polymerization mutant Bicc1 on a sucrose gradient. HEK293T cell extracts containing HA-tagged Bicc1 were fractionated on a continuous 15 to 60% sucrose gradient and analyzed by anti-Bicc1 Western blotting. The migration direction from the top to the bottom of the tube is indicated. RPS6 and γ-tubulin (γ-Tub) were used as internal controls. The graph below the gels shows the percentage of Bicc1 compared to the total Bicc1 signal for each fraction. Results represent mean values from 3 independent experiments, and error bars show SEMs. (B) Bicc1 polymer mutants fail to accumulate in cytoplasmic foci. The results of indirect immunofluorescence staining of the HA-Bicc1 WT, mutD, the ΔSAM mutant, or the bpk mutant and the P-body marker GFP-Dcp1a overexpressed in COS-1 cells are shown. Bars, 5 μm. (C) Comparative 3D rendering of the HA-Bicc1 WT and mutD by Imaris software. From the original image (center), z-stacks in two directions (z1 and z2, top and right, respectively) and 3D reconstruction (bottom) are given. Bars, 2 μm. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells and comparison with that of HA-Bicc1 mutD. Bars, 5 μm.
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Figure 5: SAM polymerization is required for Bicc1 clustering. (A) Density fractionation of WT and polymerization mutant Bicc1 on a sucrose gradient. HEK293T cell extracts containing HA-tagged Bicc1 were fractionated on a continuous 15 to 60% sucrose gradient and analyzed by anti-Bicc1 Western blotting. The migration direction from the top to the bottom of the tube is indicated. RPS6 and γ-tubulin (γ-Tub) were used as internal controls. The graph below the gels shows the percentage of Bicc1 compared to the total Bicc1 signal for each fraction. Results represent mean values from 3 independent experiments, and error bars show SEMs. (B) Bicc1 polymer mutants fail to accumulate in cytoplasmic foci. The results of indirect immunofluorescence staining of the HA-Bicc1 WT, mutD, the ΔSAM mutant, or the bpk mutant and the P-body marker GFP-Dcp1a overexpressed in COS-1 cells are shown. Bars, 5 μm. (C) Comparative 3D rendering of the HA-Bicc1 WT and mutD by Imaris software. From the original image (center), z-stacks in two directions (z1 and z2, top and right, respectively) and 3D reconstruction (bottom) are given. Bars, 2 μm. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells and comparison with that of HA-Bicc1 mutD. Bars, 5 μm.

Mentions: Among our panel of Bicc1 mutations, the mutation in mutant D disrupted the largest number of intermolecular H bonds at the SAM-SAM interface (Fig. 4C and D). To distinguish whether this interface mediates Bicc1 dimerization or the formation of higher-order assemblies, we compared the sizes of wild-type Bicc1 and mutant D in transfected HEK293T cells by sucrose gradient fractionation. Analysis of polymeric HA-Bicc1 complexes in HEK293T cells revealed a broad size distribution, with wild-type HA-Bicc1 extending beyond the fractions marked by ribosomal protein S6 (RPS6) (59), indicating that wild-type Bicc1 congregates in molecular assemblies larger than ribosomes (Fig. 5A). A similar distribution was observed for endogenous Bicc1 in extracts of mIMCD3 cells (see Fig. S4B in the supplemental material). In contrast, HA-Bicc1 mutant D was concentrated in fractions with significantly lower molecular weights in three independent experiments. These data suggest that at least the largest Bicc1 assemblies in such cell extracts likely depend on SAM polymerization. However, despite this marked shift to lower-molecular-weight fractions, HA-Bicc1 mutD levels in the first three fractions did not increase, suggesting that Bicc1 likely fails to stably accumulate as a free monomer.


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 clustering. (A) Density fractionation of WT and polymerization mutant Bicc1 on a sucrose gradient. HEK293T cell extracts containing HA-tagged Bicc1 were fractionated on a continuous 15 to 60% sucrose gradient and analyzed by anti-Bicc1 Western blotting. The migration direction from the top to the bottom of the tube is indicated. RPS6 and γ-tubulin (γ-Tub) were used as internal controls. The graph below the gels shows the percentage of Bicc1 compared to the total Bicc1 signal for each fraction. Results represent mean values from 3 independent experiments, and error bars show SEMs. (B) Bicc1 polymer mutants fail to accumulate in cytoplasmic foci. The results of indirect immunofluorescence staining of the HA-Bicc1 WT, mutD, the ΔSAM mutant, or the bpk mutant and the P-body marker GFP-Dcp1a overexpressed in COS-1 cells are shown. Bars, 5 μm. (C) Comparative 3D rendering of the HA-Bicc1 WT and mutD by Imaris software. From the original image (center), z-stacks in two directions (z1 and z2, top and right, respectively) and 3D reconstruction (bottom) are given. Bars, 2 μm. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells and comparison with that of HA-Bicc1 mutD. Bars, 5 μm.
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Figure 5: SAM polymerization is required for Bicc1 clustering. (A) Density fractionation of WT and polymerization mutant Bicc1 on a sucrose gradient. HEK293T cell extracts containing HA-tagged Bicc1 were fractionated on a continuous 15 to 60% sucrose gradient and analyzed by anti-Bicc1 Western blotting. The migration direction from the top to the bottom of the tube is indicated. RPS6 and γ-tubulin (γ-Tub) were used as internal controls. The graph below the gels shows the percentage of Bicc1 compared to the total Bicc1 signal for each fraction. Results represent mean values from 3 independent experiments, and error bars show SEMs. (B) Bicc1 polymer mutants fail to accumulate in cytoplasmic foci. The results of indirect immunofluorescence staining of the HA-Bicc1 WT, mutD, the ΔSAM mutant, or the bpk mutant and the P-body marker GFP-Dcp1a overexpressed in COS-1 cells are shown. Bars, 5 μm. (C) Comparative 3D rendering of the HA-Bicc1 WT and mutD by Imaris software. From the original image (center), z-stacks in two directions (z1 and z2, top and right, respectively) and 3D reconstruction (bottom) are given. Bars, 2 μm. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells and comparison with that of HA-Bicc1 mutD. Bars, 5 μm.
Mentions: Among our panel of Bicc1 mutations, the mutation in mutant D disrupted the largest number of intermolecular H bonds at the SAM-SAM interface (Fig. 4C and D). To distinguish whether this interface mediates Bicc1 dimerization or the formation of higher-order assemblies, we compared the sizes of wild-type Bicc1 and mutant D in transfected HEK293T cells by sucrose gradient fractionation. Analysis of polymeric HA-Bicc1 complexes in HEK293T cells revealed a broad size distribution, with wild-type HA-Bicc1 extending beyond the fractions marked by ribosomal protein S6 (RPS6) (59), indicating that wild-type Bicc1 congregates in molecular assemblies larger than ribosomes (Fig. 5A). A similar distribution was observed for endogenous Bicc1 in extracts of mIMCD3 cells (see Fig. S4B in the supplemental material). In contrast, HA-Bicc1 mutant D was concentrated in fractions with significantly lower molecular weights in three independent experiments. These data suggest that at least the largest Bicc1 assemblies in such cell extracts likely depend on SAM polymerization. However, despite this marked shift to lower-molecular-weight fractions, HA-Bicc1 mutD levels in the first three fractions did not increase, suggesting that Bicc1 likely fails to stably accumulate as a free monomer.

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