Fillebeen Carine, Wilkinson Nicole, Pantopoulos Kostas
Lady Davis Institute for Medical Research, Jewish General Hospital; Department of Medicine, McGill University.
Lady Davis Institute for Medical Research, Jewish General Hospital; Department of Medicine, McGill University;
J Vis Exp. 2014 Dec 3(94):52230. doi: 10.3791/52230.
RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of(32) P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called "gel retardation" or "bandshift" assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.
RNA/蛋白质相互作用对于转录后调控途径至关重要。在特征最明确的胞质RNA结合蛋白中，铁调节蛋白IRP1和IRP2最为突出。它们与几种靶mRNA的非翻译区（UTR）内的铁反应元件（IRE）结合，从而控制mRNA的翻译或稳定性。IRE/IRP相互作用已通过电泳迁移率变动分析（EMSA）进行了广泛研究。在此，我们描述了用于分析IRP1和IRP2的IRE结合活性的EMSA方案，该方案也可推广用于评估其他RNA结合蛋白的活性。将含有RNA结合蛋白的粗蛋白裂解物或该蛋白的纯化制剂与过量的（32）P标记的RNA探针孵育，以形成复合物。加入肝素以防止非特异性的蛋白质与探针结合。随后，通过在聚丙烯酰胺凝胶上进行非变性电泳分析混合物。游离探针迁移速度快，而RNA/蛋白质复合物迁移速度慢；因此，该方法也称为“凝胶阻滞”或“条带迁移”分析。电泳完成后，将凝胶干燥，并通过放射自显影检测RNA/蛋白质复合物以及游离探针。该方案的总体目标是检测和定量IRE/IRP以及其他RNA/蛋白质相互作用。此外，EMSA还可用于确定所研究的RNA/蛋白质相互作用的特异性、结合亲和力和化学计量。
