Methods to Study Protein-Binding to Pseudogene Transcripts.

One of the most commonly described biological feature of processed pseudogenes is the ability to influence the expression of their parental coding genes. As evidenced in several studies, the high sequence similarity between these RNA pairs sets up a certain level of competition for posttranscriptional regulators, including, among others, RNA-binding proteins (RBPs). RBPs may affect, positively or negatively, the stability of bound mRNAs, so that, if an overexpressed pseudogene competes with its homologous coding gene, the downstream protein synthesis would change, with potential pathological consequences. Given these premises, a rigorous and comprehensive understanding of interactions between pseudogene-parental gene RNA pairs and RBPs could provide further insights into the biological bases of complex diseases, such as cancer, cardiovascular disease, and type 2 diabetes, identifying novel predictive and/or prognostic biomarkers.Herein, we detail easily adaptable protocols of plasmid-based molecular cloning and RNA-electrophoretic mobility shift assay (EMSA) used in our laboratory for determining the interaction between a cytoplasmatic stabilizing protein (αCP1) and the pseudogene-parental gene RNA pair HMGA1-p /HMGA1. We also offer a general overview of RNA immunoprecipitation procedures and present novel bioinformatic tools for predicting RBPs binding sites on pseudogene transcripts.