Abstract：
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.
摘要：
加工假基因最常描述的生物学特征之一是影响其亲本编码基因表达的能力。正如几项研究所证明的那样，这些RNA对之间的高度序列相似性为转录后调节因子建立了一定水平的竞争，包括,其中,RNA结合蛋白(RBP)。RBP可能会影响，积极或消极，结合的mRNA的稳定性，所以，如果过表达的假基因与其同源编码基因竞争，下游的蛋白质合成会发生变化，具有潜在的病理后果。鉴于这些前提,对假基因-亲本基因RNA对和RBPs之间的相互作用的严格和全面的理解可以为复杂疾病的生物学基础提供进一步的见解，比如癌症，心血管疾病,和2型糖尿病，识别新的预测和/或预后生物标志物。在这里，我们详细介绍了基于质粒的分子克隆和RNA电泳迁移率变化分析(EMSA)的易于适应的方案,这些方案在我们的实验室中用于确定细胞质稳定蛋白(αCP1)和假基因-亲本RNA对HMGA1-p/HMGA1之间的相互作用.我们还提供了RNA免疫沉淀程序的一般概述，并提出了用于预测假基因转录物上RBP结合位点的新型生物信息学工具。
