Bicc1 Polymerization Regulates the Localization and Silencing of Bound mRNA.
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.
Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.Show MeSH
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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.
Affiliation: Ecole Polytechnique Fédérale de Lausanne (EPFL), SV ISREC, Lausanne, Switzerland.