Limits...
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.

Show MeSH

Related in: MedlinePlus

Bicc1 concentrates an associated reporter mRNA in cytoplasmic foci. (A) Principle of the MS2-YFP colocalization assay. The 3′ extremity of the Luc-AC6 reporter mRNA was fused to 27 MS2 hairpins, which constitute multiple binding sites for the MS2 protein fused to YFP. CDS, coding sequence; prox, proximal. (B) RNA coimmunoprecipitation. The Luc-AC6-MS2×27 reporter mRNA was expressed in HEK293T cells together with HA-Bicc1 or empty vector. After HA immunoprecipitation (IP), the various fractions were analyzed by Western blotting and by RT-PCR. Five percent of the total extract was used as the input. The β-actin mRNA was used as a negative control for the RT-PCR. HA-Bicc1 lacking all KH and KH-like domains (ΔKH) was used as an additional negative control for RNA-binding specificity. (C) Cotransfection of the fluorescent YFP-MS2 fusion protein does not affect the silencing of the Luc-AC6-MS2×27 reporter by HA-Bicc1. β-Galactosidase was used as a control for normalization, and the data represent the percent expression relative to that of a mock-treated control. Error bars show SEMs. *, P < 0.005. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells. The MS2-tagged mRNA was detected by the relocalization of fluorescent MS2-YFP fusion protein, which binds the MS2 RNA hairpins. Bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4561730&req=5

Figure 2: Bicc1 concentrates an associated reporter mRNA in cytoplasmic foci. (A) Principle of the MS2-YFP colocalization assay. The 3′ extremity of the Luc-AC6 reporter mRNA was fused to 27 MS2 hairpins, which constitute multiple binding sites for the MS2 protein fused to YFP. CDS, coding sequence; prox, proximal. (B) RNA coimmunoprecipitation. The Luc-AC6-MS2×27 reporter mRNA was expressed in HEK293T cells together with HA-Bicc1 or empty vector. After HA immunoprecipitation (IP), the various fractions were analyzed by Western blotting and by RT-PCR. Five percent of the total extract was used as the input. The β-actin mRNA was used as a negative control for the RT-PCR. HA-Bicc1 lacking all KH and KH-like domains (ΔKH) was used as an additional negative control for RNA-binding specificity. (C) Cotransfection of the fluorescent YFP-MS2 fusion protein does not affect the silencing of the Luc-AC6-MS2×27 reporter by HA-Bicc1. β-Galactosidase was used as a control for normalization, and the data represent the percent expression relative to that of a mock-treated control. Error bars show SEMs. *, P < 0.005. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells. The MS2-tagged mRNA was detected by the relocalization of fluorescent MS2-YFP fusion protein, which binds the MS2 RNA hairpins. Bars, 5 μm.

Mentions: Bicc1 clustering may influence the localization of associated target mRNAs or affect RNA binding, or both. To distinguish between these possibilities, we monitored the localization of a reporter mRNA containing the Bicc1 binding region of the AC6 3′ UTR (27) and 27 repeats of a motif that binds the bacteriophage MS2 coat protein (the MS2×27 motif) (55). Coimmunoprecipitation and luciferase assays with HA-Bicc1 from extracts of transfected HEK293T cells showed that Bicc1 specifically binds this reporter mRNA and inhibits its expression, as expected (Fig. 2A to C). To assess whether Bicc1 influences mRNA localization, we cotransfected a fusion protein of MS2 with nuclear yellow fluorescent protein (YFP) into COS-1 cells. Like HEK293T cells, COS-1 cells show no endogenous Bicc1 expression but are more adhesive, have a larger cytoplasm, and, thus, are more amenable for imaging. MS2-YFP without Luc-AC6-MS2×27 reporter mRNA localized to nuclei both in the presence and in the absence of HA-Bicc1 (Fig. 2D, rows 1 and 2). However, when coexpressed with Luc-AC6-MS2×27 and without Bicc1, MS2-YFP accumulated diffusely throughout the cytoplasm, confirming that MS2-YFP was exported from the nucleus together with the reporter mRNA (Fig. 2D, row 3). Interestingly, the combined expression of the Luc-AC6-MS2×27 reporter with HA-Bicc1 led to the concentration of MS2-YFP in cytoplasmic Bicc1 puncta, with 89% ± 8% of the cytoplasmic YFP signal colocalizing with Bicc1 foci (Fig. 2D, row 4). We therefore asked whether the Luc-AC6-MS2×27 reporter also enters cytoplasmic foci in the mIMCD3 mouse inner medullary collecting duct cell line that expresses endogenous Bicc1 (56). Transfection of the reporter RNA was sufficient to translocate the MS2-YFP fusion protein from the nucleus to cytoplasmic Bicc1-stained foci independently of exogenous Bicc1 (see Fig. S4A in the supplemental material). Even though we could not sufficiently deplete endogenous Bicc1 foci by RNA interference to more conclusively validate their function (not shown), their colocalization with reporter RNA strongly corroborates our conclusion that target mRNAs are recruited to endogenous Bicc1 in cytoplasmic clusters.


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)

Bicc1 concentrates an associated reporter mRNA in cytoplasmic foci. (A) Principle of the MS2-YFP colocalization assay. The 3′ extremity of the Luc-AC6 reporter mRNA was fused to 27 MS2 hairpins, which constitute multiple binding sites for the MS2 protein fused to YFP. CDS, coding sequence; prox, proximal. (B) RNA coimmunoprecipitation. The Luc-AC6-MS2×27 reporter mRNA was expressed in HEK293T cells together with HA-Bicc1 or empty vector. After HA immunoprecipitation (IP), the various fractions were analyzed by Western blotting and by RT-PCR. Five percent of the total extract was used as the input. The β-actin mRNA was used as a negative control for the RT-PCR. HA-Bicc1 lacking all KH and KH-like domains (ΔKH) was used as an additional negative control for RNA-binding specificity. (C) Cotransfection of the fluorescent YFP-MS2 fusion protein does not affect the silencing of the Luc-AC6-MS2×27 reporter by HA-Bicc1. β-Galactosidase was used as a control for normalization, and the data represent the percent expression relative to that of a mock-treated control. Error bars show SEMs. *, P < 0.005. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells. The MS2-tagged mRNA was detected by the relocalization of fluorescent MS2-YFP fusion protein, which binds the MS2 RNA hairpins. Bars, 5 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4561730&req=5

Figure 2: Bicc1 concentrates an associated reporter mRNA in cytoplasmic foci. (A) Principle of the MS2-YFP colocalization assay. The 3′ extremity of the Luc-AC6 reporter mRNA was fused to 27 MS2 hairpins, which constitute multiple binding sites for the MS2 protein fused to YFP. CDS, coding sequence; prox, proximal. (B) RNA coimmunoprecipitation. The Luc-AC6-MS2×27 reporter mRNA was expressed in HEK293T cells together with HA-Bicc1 or empty vector. After HA immunoprecipitation (IP), the various fractions were analyzed by Western blotting and by RT-PCR. Five percent of the total extract was used as the input. The β-actin mRNA was used as a negative control for the RT-PCR. HA-Bicc1 lacking all KH and KH-like domains (ΔKH) was used as an additional negative control for RNA-binding specificity. (C) Cotransfection of the fluorescent YFP-MS2 fusion protein does not affect the silencing of the Luc-AC6-MS2×27 reporter by HA-Bicc1. β-Galactosidase was used as a control for normalization, and the data represent the percent expression relative to that of a mock-treated control. Error bars show SEMs. *, P < 0.005. (D) Localization by indirect immunofluorescence staining of the Luc-AC6-MS2×27 reporter mRNA and HA-Bicc1 in COS-1 cells. The MS2-tagged mRNA was detected by the relocalization of fluorescent MS2-YFP fusion protein, which binds the MS2 RNA hairpins. Bars, 5 μm.
Mentions: Bicc1 clustering may influence the localization of associated target mRNAs or affect RNA binding, or both. To distinguish between these possibilities, we monitored the localization of a reporter mRNA containing the Bicc1 binding region of the AC6 3′ UTR (27) and 27 repeats of a motif that binds the bacteriophage MS2 coat protein (the MS2×27 motif) (55). Coimmunoprecipitation and luciferase assays with HA-Bicc1 from extracts of transfected HEK293T cells showed that Bicc1 specifically binds this reporter mRNA and inhibits its expression, as expected (Fig. 2A to C). To assess whether Bicc1 influences mRNA localization, we cotransfected a fusion protein of MS2 with nuclear yellow fluorescent protein (YFP) into COS-1 cells. Like HEK293T cells, COS-1 cells show no endogenous Bicc1 expression but are more adhesive, have a larger cytoplasm, and, thus, are more amenable for imaging. MS2-YFP without Luc-AC6-MS2×27 reporter mRNA localized to nuclei both in the presence and in the absence of HA-Bicc1 (Fig. 2D, rows 1 and 2). However, when coexpressed with Luc-AC6-MS2×27 and without Bicc1, MS2-YFP accumulated diffusely throughout the cytoplasm, confirming that MS2-YFP was exported from the nucleus together with the reporter mRNA (Fig. 2D, row 3). Interestingly, the combined expression of the Luc-AC6-MS2×27 reporter with HA-Bicc1 led to the concentration of MS2-YFP in cytoplasmic Bicc1 puncta, with 89% ± 8% of the cytoplasmic YFP signal colocalizing with Bicc1 foci (Fig. 2D, row 4). We therefore asked whether the Luc-AC6-MS2×27 reporter also enters cytoplasmic foci in the mIMCD3 mouse inner medullary collecting duct cell line that expresses endogenous Bicc1 (56). Transfection of the reporter RNA was sufficient to translocate the MS2-YFP fusion protein from the nucleus to cytoplasmic Bicc1-stained foci independently of exogenous Bicc1 (see Fig. S4A in the supplemental material). Even though we could not sufficiently deplete endogenous Bicc1 foci by RNA interference to more conclusively validate their function (not shown), their colocalization with reporter RNA strongly corroborates our conclusion that target mRNAs are recruited to endogenous Bicc1 in cytoplasmic clusters.

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