title: A versatile assay for RNA-binding proteins in living cells.
abstract: RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.
authors.forename: Claudia
authors.initials: C
authors.lastname: Strein
authors.forename: Anne-Marie
authors.initials: AM
authors.lastname: Alleaume
authors.forename: Ulrich
authors.initials: U
authors.lastname: Rothbauer
authors.forename: Matthias W
authors.initials: MW
authors.lastname: Hentze
authors.forename: Alfredo
authors.initials: A
authors.lastname: Castello
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry; D006367:HeLa Cells; D022781:Hepatocytes / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D011485:Protein Binding; D012313:RNA / Q000096:biosynthesis*; D020871:RNA Stability / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics*
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D012313:RNA
keywords: RNA; RNA-binding proteins; fluorescence; protein–RNA interaction