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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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Impaired localization of Bicc1 in bpk mutant mouse kidney. (A) Frozen sections of WT and bpk mutant mouse kidneys labeled with anti-Bicc1 antibodies and with the proximal tubule marker LTL on postnatal day 11. Boxed areas are magnified in the panels to the right. Bars, 20 μm (large views) and 10 μm (magnified views). (B) Bicc1 isoform B (Bicc1-B) accumulates in cytoplasmic foci. The results of indirect immunofluorescence staining of HA-Bicc1 isoforms A and B and the P-body marker GFP-Dcp1a in transfected COS-1 cells are shown. Bars, 5 μm. (C) Silencing of AC6 3′ UTR luciferase reporter by HA-Bicc1 isoform A or B in HEK293T cells. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
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Figure 7: Impaired localization of Bicc1 in bpk mutant mouse kidney. (A) Frozen sections of WT and bpk mutant mouse kidneys labeled with anti-Bicc1 antibodies and with the proximal tubule marker LTL on postnatal day 11. Boxed areas are magnified in the panels to the right. Bars, 20 μm (large views) and 10 μm (magnified views). (B) Bicc1 isoform B (Bicc1-B) accumulates in cytoplasmic foci. The results of indirect immunofluorescence staining of HA-Bicc1 isoforms A and B and the P-body marker GFP-Dcp1a in transfected COS-1 cells are shown. Bars, 5 μm. (C) Silencing of AC6 3′ UTR luciferase reporter by HA-Bicc1 isoform A or B in HEK293T cells. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.

Mentions: To validate a potential influence of the bpk mutation on polymeric Bicc1 in vivo, cryosections of kidneys obtained from bpk mice postnatally were colabeled using anti-Bicc1 antibody and LTL to mark proximal tubules. Compared to the findings for wild-type Bicc1, where Bicc1 was concentrated in cytoplasmic foci of both LTL-positive and LTL-negative renal tubule cells, the cystic kidneys of bpk mutant animals showed considerably weaker and more diffuse Bicc1 staining that was confined to LTL-positive structures (Fig. 7A). This result concurs with the effects of bpk on Bicc1 protein stability and cytoplasmic clustering observed ex vivo. However, despite the abnormally diffuse distribution, some Bicc1 foci remained clearly detectable. To evaluate whether these residual foci could arise from the alternatively spliced transcript B that is unaffected by the bpk mutation (17, 22) (see Fig. S1C in the supplemental material), we overexpressed a synthetic cDNA of this splice variant in transfected cells. Similar to the findings for full-length Bicc1 encoded by transcript A, transfected isoform B was readily detected by anti-Bicc1 antibody in cytoplasmic foci, and it efficiently silenced the Luc-AC6 reporter mRNA (Fig. 7B and C). It is likely, therefore, that the bpk mutant phenotype is caused at least in part by the failure of the affected Bicc1 isoform A to polymerize. In addition, these results are consistent with the notion that isoform B remains functional and thus likely accounts for residual hypomorphic Bicc1 activity in bpk mutants. Alternatively, or in addition, cystic growth in bpk mutants may also be attenuated by polymerization-independent Bicc1 functions, e.g., during Wnt signaling.


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)

Impaired localization of Bicc1 in bpk mutant mouse kidney. (A) Frozen sections of WT and bpk mutant mouse kidneys labeled with anti-Bicc1 antibodies and with the proximal tubule marker LTL on postnatal day 11. Boxed areas are magnified in the panels to the right. Bars, 20 μm (large views) and 10 μm (magnified views). (B) Bicc1 isoform B (Bicc1-B) accumulates in cytoplasmic foci. The results of indirect immunofluorescence staining of HA-Bicc1 isoforms A and B and the P-body marker GFP-Dcp1a in transfected COS-1 cells are shown. Bars, 5 μm. (C) Silencing of AC6 3′ UTR luciferase reporter by HA-Bicc1 isoform A or B in HEK293T cells. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
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Figure 7: Impaired localization of Bicc1 in bpk mutant mouse kidney. (A) Frozen sections of WT and bpk mutant mouse kidneys labeled with anti-Bicc1 antibodies and with the proximal tubule marker LTL on postnatal day 11. Boxed areas are magnified in the panels to the right. Bars, 20 μm (large views) and 10 μm (magnified views). (B) Bicc1 isoform B (Bicc1-B) accumulates in cytoplasmic foci. The results of indirect immunofluorescence staining of HA-Bicc1 isoforms A and B and the P-body marker GFP-Dcp1a in transfected COS-1 cells are shown. Bars, 5 μm. (C) Silencing of AC6 3′ UTR luciferase reporter by HA-Bicc1 isoform A or B in HEK293T cells. β-Galactosidase was cotransfected for signal normalization. Error bars show SEMs. *, P < 0.005.
Mentions: To validate a potential influence of the bpk mutation on polymeric Bicc1 in vivo, cryosections of kidneys obtained from bpk mice postnatally were colabeled using anti-Bicc1 antibody and LTL to mark proximal tubules. Compared to the findings for wild-type Bicc1, where Bicc1 was concentrated in cytoplasmic foci of both LTL-positive and LTL-negative renal tubule cells, the cystic kidneys of bpk mutant animals showed considerably weaker and more diffuse Bicc1 staining that was confined to LTL-positive structures (Fig. 7A). This result concurs with the effects of bpk on Bicc1 protein stability and cytoplasmic clustering observed ex vivo. However, despite the abnormally diffuse distribution, some Bicc1 foci remained clearly detectable. To evaluate whether these residual foci could arise from the alternatively spliced transcript B that is unaffected by the bpk mutation (17, 22) (see Fig. S1C in the supplemental material), we overexpressed a synthetic cDNA of this splice variant in transfected cells. Similar to the findings for full-length Bicc1 encoded by transcript A, transfected isoform B was readily detected by anti-Bicc1 antibody in cytoplasmic foci, and it efficiently silenced the Luc-AC6 reporter mRNA (Fig. 7B and C). It is likely, therefore, that the bpk mutant phenotype is caused at least in part by the failure of the affected Bicc1 isoform A to polymerize. In addition, these results are consistent with the notion that isoform B remains functional and thus likely accounts for residual hypomorphic Bicc1 activity in bpk mutants. Alternatively, or in addition, cystic growth in bpk mutants may also be attenuated by polymerization-independent Bicc1 functions, e.g., during Wnt signaling.

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