Abstract
Bicaudal-C (Bic-C) RNA binding proteins function as important translational repressors in multiple biological contexts within metazoans. However, their RNA binding sites are unknown. We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development. This repression contributes to normal development by confining the xCR1 protein, a regulator of key signaling pathways, to specific cells of the embryo. In this report, we combined biochemical approaches with in vivo mRNA reporter assays to define the minimal Bic-C target site within the xCR1 mRNA. This 32-nucleotide Bic-C target site is predicted to fold into a stem-loop secondary structure. Mutational analyses provided evidence that this stem-loop structure is important for Bic-C binding. The Bic-C target site was sufficient for Bic-C mediated repression in vivo. Thus, we describe the first RNA binding site for a Bic-C protein. This identification provides an important step toward understanding the mechanisms by which evolutionarily conserved Bic-C proteins control cellular function in metazoans.
Highlights
Bicaudal-C RNA binding proteins are important translational repressors in many different biological contexts
We recently demonstrated that Bic-C functions in spatially regulated translational repression of the xCR1 mRNA during Xenopus development
We identified a region of the xCR1 mRNA 3Ј-UTR that was necessary and sufficient for Bic-C binding to RNA as well as Bic-Cdependent translational repression
Summary
Bicaudal-C RNA binding proteins are important translational repressors in many different biological contexts. Bic-C mutant mice exhibit defects in the normal left/right patterning of the internal organs and develop renal cysts [4, 5], and mutations in the human Bic-C gene are associated with a form of kidney disease called cystic renal dysplasia [6] These examples illustrate the biological importance of Bic-C proteins, the RNA binding site(s) for Bic-Cs from any organism have not been described. We used EMSA, RNase protection, and RNA footprinting assays to define a 32-nucleotide region within the xCR1 TCE that functions as a minimal Bic-C binding site This site is predicted to form a stem-loop secondary structure, and mutational analyses supported the importance of this secondary structure for Bic-C-RNA interactions. We have defined the first Bic-C target site that is functional for both specific Bic-C binding in vitro and Bic-C-mediated translational repression in vivo
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