Optimized RNA gel-shift and UV cross-linking assays for characterization of cytoplasmic RNA-protein interactions

Considerable interest has recently focused on defining the mechanisms invol ved in the regulation of gene expression at the level of mRNA stability and translational efficiency. However the assays used to directly investigate interactions between RNA and cytoplasmic proteins have been difficult to es tablish, and methods are nor widely available. Here, we describe a robust m ethod for RNA electrophoretic mobility shift and UV cross-linking assays th at allows rapid detection of cytoplasmic RNA-protein interactions. For adde d convenience to new investigators, these assays use mini-gels with an elec trophoresis time of 15-20 min, enabling a high throughput of samples. The m ethod works successfully with many different probes and cytoplasmic extract s from a variety of cell lines. Furthermore, we provide a system to optimiz e characterization of the RNA-protein complex and troubleshoot most assay d ifficulties.