RNAcompete methodology and application to determine sequence preferences of unconventional RNA-binding proteins

RNA-binding proteins (RBPs) participate in diverse cellular processes and have important roles in human development and disease. The human genome, and that of many other eukaryotes, encodes hundreds of RBPs that contain canonical sequence-specific RNA-binding domains (RBDs) as well as numerous other unconventional RNA binding proteins (ucRBPs). ucRBPs physically associate with RNA but lack common RBDs. The degree to which these proteins bind RNA, in a sequence specific manner, is unknown. Here, we provide a detailed description of both the laboratory and data processing methods for RNAcompete, a method we have previously used to analyze the RNA binding preferences of hundreds of RBD-containing RBPs, from diverse eukaryotes. We also determine the RNA-binding preferences for two human ucRBPs, NUDT21 and CNBP, and use this analysis to exemplify the RNAcompete pipeline. The results of our RNAcompete experiments are consistent with independent RNA-binding data for these proteins and demonstrate the utility of RNAcompete for analyzing the growing repertoire of ucRBPs.