Enhanced Immunoprecipitation Techniques for the Identification of RNA Binding Protein Partners: CRD-BP interactions in mammary epithelial cells

Abstract

AbstractRNA binding proteins (RBPs) regulate expression of large cohorts of RNA species to affect programmatic changes in cellular phenotypes. In order to describe the function of RBPs within a cell, it is key to identify their mRNA binding partners. This is often done by cross-linking nucleic acids to RBPs, followed by chemical release of the nucleic acid fragments for analysis. However, this methodology is lengthy, involves complex processing leading to extraordinary losses, requires large amounts of starting materials, and is prone to artifacts due to the labile nature of mRNA. To evaluate potential alternative technologies, we tested “exclusion-based” purification of immunoprecipitates (oil-based IFAST™ or air-based SLIDE™), and report here that these methods can efficiently, rapidly and specifically isolate RBP-RNA complexes with minimal handling. The analysis starts with >100x less material than for techniques that include cross-linking. Depending on the specific antibody used, 50-100% of starting protein is retrieved, allowing the assay of endogenous levels of RBP instead of tagged and over-expressed ectopic proteins. Isolated protein and nucleic acid components are purified and analyzed using standard techniques to provide a comprehensive portrait of RBP complexes. Using exclusion-based techniques, we show that the mRNA binding partners for CRD-BP/IMP1/IGF2BP1/ZBP1 in cultured mammary epithelial cells are enriched in mRNAs important for de-toxifying superoxides (glutathione metabolic enzymes) and other mRNAs encoding mitochondrial proteins.