A Methodology To Separate And Identify RNA-binding Protein-Associated Ribonucleoprotein Complex In Vivo

Posttranscriptional gene expression control is important to cellular growth and differentiation of higher eukaryotes, as it plays a pivotal role in determining the eventual expression level and cellular localization of many proteins. RNA —binding proteins (RBPs), which usually have conserved RNA-binding motifs, are key molecules in posttranscriptional gene expression control. These conserved RNA-binding motifs can specifically interact with target RNAs through specific sequences and structures to regulate splicing, stability, translation efficiency and subcellular transportation of RNAs. In post-genomic era, how to efficiently recover and identify the ribonucleoproteins of RNA-binding proteins in vivo is a significant challenge.We present here a methodology to separate, identify and profile the target RNAs of RNA-binding proteins. Firstly, we subcloned biotin ligase (BirA) and RNA-binding protein with biotin acceptor peptide into mammalian expression vectors. Then, we co-transfected them into cells in the existence of biotin. Since BirA can biotinylate the biotin acceptor peptide tags, Streptavidin Sepharose-beads were used to single-purified and recovered the ribonucleoprotein (RNP) complex, which contains the RNA binding protein and their target RNAs. Furthermore, by using Western blot, we validated the reproduction of biotinylation and the efficiency of the single-purification through the Streptavidin Sepharose-beads. Finally, by using RT-PCR, we validated the known target RNAs of the ribonucleoprotein complex of PCBP2, which targets and regulates the mRNAs of Trx2, Cln2, PPT2 and coxll. Thus, we here provided a dependable and efficient methodology to explore other novel RNA binding proteins in cultured eukaryote cells.