RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not\nreflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their\nresponses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results\nserve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to\nanalyze proteinââ?¬â??mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed\nin cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain\nantibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins\nis assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to\nliving cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or\npharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available\nGBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment.\nTherefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast\nand simple radioactivity-free method will find many useful applications in RNA biology.
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