RNA targets and cellular function profiling of the RNA-binding protein Bicaudal C homolog 1 (BICC1)

Abstract

The Bicaudal C homolog 1 (BICC1) is a RNA-binding protein characterized by three K Homology domains in the N terminal end (KH1, KH2 and KH3 domains) which are responsible for the RNA-binding activity, and a Sterile Alpha Motif at the C-terminal end (SAM domain) which was related to the promotion of protein-protein interaction. The protein has been studied by its role in polycystic kidney disease (PKD) and kidney organogenesis, acting in the main pathways involved with this process. Even with many studies, the overall group of mRNA binding targets of the BICC1 protein are unknow. The understanding of mRNA targets is crucial to reveal BICC1 binding mode and its cellular function. In this project we propose a set of experiments to uncover the RNAs targets of BICC1 and also analyze the role of the KH domains for the BICC1 function. To discovery the BICC1 RNA-targets we will overexpress the FLAG-fusion of the protein in cell and perform the RNA Immunoprecipitation (RIPseq), a procedure where the target protein is immunoprecipitated followed by isolation of its bound RNA targets, which are then sequenced. To investigate the importance of the KH domains in the BICC1 binding to RNAs, we will also mutate the protein construct by site directed mutagenesis and compare to the wild type RIPseq results. In parallel we will monitor the subcellular localization of BICC1 and its mutated forms by immunofluorescence, considering that a few publications have shown a link with cytoplasmic stress granules. The combined study of the protein and its mutant forms for RNA binding will help to elucidate BICC1 function on RNA regulation.