Bicaudal-C spatially controls translation of vertebrate maternal mRNAs

Abstract

The Xenopus Cripto-1 protein is confined to the cells of the animal hemisphere during early embryogenesis where it regulates the formation of anterior structures. Cripto-1 protein accumulates only in animal cells because cripto-1 mRNA in cells of the vegetal hemisphere is translationally repressed. Here, we show that the RNA binding protein, Bicaudal-C (Bic-C), functioned directly in this vegetal cell-specific repression. While Bic-C protein is normally confined to vegetal cells, ectopic expression of Bic-C in animal cells repressed a cripto-1 mRNA reporter and associated with endogenous cripto-1 mRNA. Repression by Bic-C required its N-terminal domain, comprised of multiple KH motifs, for specific binding to relevant control elements within the cripto-1 mRNA and a functionally separable C-terminal translation repression domain. Bic-C-mediated repression required the 5 ′ CAP and translation initiation factors, but not a poly(A) tail or the conserved SAM domain within Bic-C. Bic-C-directed immunoprecipitation followed by deep sequencing of associated mRNAs identified multiple Bic-C-regulated mRNA targets, including cripto-1 mRNA, providing new insights and tools for understanding the role of Bic-C in vertebrate development.

A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators

“…The reporter mRNA containing the wnt11b 3′UTR was repressed by co-expression of HA-Bicc1 to the same extent as the reporter containing the cripto1 3′UTR that we previously identified and characterized as a Bicc1-regulated mRNA (Fig. 3C) (Zhang et al, 2009(Zhang et al, , 2013. The effect was specific, as a reporter mRNA containing the 3′UTR from the cyclin B1 (ccnb1) mRNA, an mRNA not regulated by Bicc1, was not repressed.…”

“…This phenotype showed similarity to that described for embryos that overexpress the Wnt11b ligand (Tao et al, 2005), raising the possibility that wnt11b mRNA was a direct or indirect target of Bicc1 translational repression. Although wnt11b mRNA was not identified as a Bicc1 target in our previous studies using an ectopically expressed HA-Bicc1 immunoprecipitation strategy that identified several mRNAs relevant to cell-fate decisions, there were several possible technical reasons that could cause relevant direct targets to be missed (Zhang et al, 2013). Therefore, we tested whether wnt11b mRNA encoded elements sufficient to allow Bicc1-mediated repression using a luciferase reporter mRNA assay described in our previous work (Zhang et al, 2013).…”

“…Although wnt11b mRNA was not identified as a Bicc1 target in our previous studies using an ectopically expressed HA-Bicc1 immunoprecipitation strategy that identified several mRNAs relevant to cell-fate decisions, there were several possible technical reasons that could cause relevant direct targets to be missed (Zhang et al, 2013). Therefore, we tested whether wnt11b mRNA encoded elements sufficient to allow Bicc1-mediated repression using a luciferase reporter mRNA assay described in our previous work (Zhang et al, 2013). Specifically, an mRNA containing the luciferase coding region fused to the X. laevis wnt11b mRNA 3′UTR was injected into animal cells of 4-to 8-cell embryos, with and without a synthetic mRNA encoding HA-tagged Bicc1 (HA-Bicc1) (Fig.…”

“…However, only a few studies present data linking Bicc1-dependent translation repression of relevant target mRNAs to its biological roles (Chicoine et al, 2007;Piazzon et al, 2012;Zhang et al, 2013). In recent studies of maternally controlled stages of embryogenesis of Xenopus laevis, we have presented evidence that several maternal mRNAs encoding important cell-fate regulators can bind Bicc1 and are potential targets of Bicc1-mediated translational repression, including most notably the cripto1 (tdgf1.3 -Xenbase) mRNA for which we have defined a Bicc1 RNA-binding element in the 3′ untranslated region (3′UTR) (Zhang et al, 2013). However, although these data are consistent with a model in which the translational repression activity of Bicc1 regulates the earliest steps of vertebrate embryogenesis, such as establishing the body axes, experiments that directly address the role of maternal Bicc1 in vertebrates are needed to test this model and establish a connection between the biological and biochemical roles of Bicc1.…”

“…The Bicc1-depleted embryos exhibited greatly reduced levels of Bicc1 protein, developed with an excess of dorsal-anterior structures and overexpressed several genes that define Spemann's organizer, the organizing tissue that controls cell-fate decisions after zygotic transcription begins (reviewed by Houston, 2012). In previous studies, we identified the cripto1 and coco (dand5 -Xenbase) mRNAs as potential targets of Bicc1-mediated repression (Zhang et al, 2013). These data along with previous phenotypic studies of cripto1 and coco mutants help link the biological role of Bicc1 in embryogenesis documented here to its biochemical role in translational repression (Tao et al, 2005;Bates et al, 2013).…”