Defining the molecular determinants required for RNA binding functions of the Bicaudal-C (Bicc1) translational repressor protein

Abstract

Bicaudal-C (Bicc1) is an RNA binding protein that functions as translational repressor in all animal species. Its repression activities are required for a multitude of biological processes such as guiding the earliest steps of embryonic development and organ homeostasis. For example, defects in Bicc1 expression or its activities can give rise to polycystic kidney disease in humans, a disorder that is characterized by an abnormal collection of cysts in the kidney. In addition, Bicc1 functions in vertebrate embryos to control the translation of specific maternal mRNAs that encode key developmental regulatory proteins. During the early stages of vertebrate development defects in Bicc1 trigger cell fate changes that lead to embryonic malformations similar to some human birth defects. A general feature is that in all of its biological contexts Bicc1 binds to specific mRNAs and it is through this binding that it selects specific mRNAs for translational repression. My project is focused on defining the molecular determinants of the Bicc1 protein required for that specific mRNA binding. Recent results from the Sheets lab identified a 70 amino acid region of the Bicc1 protein, the KH2 domain that is required for mRNA binding. However, the particular molecular features within the KH2 domain important for RNA binding activity are unknown. To address this issue, I am generating mutations of the KH2 domain that change evolutionarily conserved amino acid residues in all Bicc1 proteins. These mutant proteins will be analyzed with gel shift RNA binding assays in vitro as well as in vivo RNA binding experiments performed in Xenopus embryos. My goal is to define the amino acid residues in the KH2 domain that are responsible for providing specificity for Bicc1 binding to specific mRNAs, guide its repression activities and mediate its biological functions.