Evaluation of post-transcriptional gene regulation in pancreatic cancer cells: Studying RNA binding proteins and their mRNA targets

Post-transcriptional regulation of gene expression through interaction between RNA binding proteins (RBPs) and target mRNAs have gained considerable interest over the last decade. Altered expression of RBPs as detected in pancreatic ductal adenocarcinoma (PDAC) cells alters mRNA processing, and in turn, the entire transcriptome and proteome. Thus, this gene regulatory mechanism can regulate important pro-oncogenic signaling pathways (e.g., TP53, WEE1, and c-MYC) in PDAC cells. Ribonucleoprotein immunoprecipitation assays (RNP-IP or RIP) are a modified immunoprecipitation method to study physical interactions between RBPs and their mRNA targets. As a first step to explore RBP interactomes and define novel therapeutic targets and dysregulated pathways in disease, RIPs are a sensitive and established molecular biology technique used to isolate and differentiate bound transcripts to RBPs in a variety of experimental conditions. This chapter describes an up-to-date, detailed protocol for performing this assay in mammalian cytoplasmic extracts (i.e., PDAC cells), and reviews current methods to validate target binding sites such as electrophoretic mobility shift assay (EMSA) and cross-linking immunoprecipitation polymerase chain reaction (CLIP-PCR).