title: Identification of mRNA-Interacting Factors by MS2-TRAP (MS2-Tagged RNA Affinity Purification).
abstract: Posttranscriptional gene expression is governed by the interaction of mRNAs with vast families of RNA-binding proteins (RBPs) and noncoding (nc)RNAs. RBPs and ncRNAs jointly influence all aspects of posttranscriptional metabolism, including pre-mRNA splicing and maturation, mRNA transport, editing, stability, and translation. Given the impact of mRNA-interacting molecules on gene expression, there is great interest in identifying mRNA-binding factors comprehensively. Here, we provide a detailed protocol to tag mRNAs with MS2 hairpins and then affinity-purify trans-binding factors (RBPs, ncRNAs) associated with the MS2-tagged mRNA. This method, termed MS2-TRAP, permits the systematic characterization of ribonucleoprotein (RNP) complexes formed on a given mRNA of interest. We describe how to prepare the mRNA-MS2 expression vector, purify the MS2-tagged RNP complexes, and detect bound RNAs and RBPs, as well as variations of this methodology to address related questions of RNP biology.
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, 173 Ashley Avenue, Charleston, SC, 29425, USA. yoonje@musc.edu.
authors.forename: Je-Hyun
authors.initials: JH
authors.lastname: Yoon
authors.affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Laboratory of Genetics and Genomics, National Institute on Aging-Intramural Research Program, National Institutes of Health, 173 Ashley Avenue, Charleston, SC, 29425, USA.
authors.forename: Myriam
authors.initials: M
authors.lastname: Gorospe
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002846:Chromatography, Affinity / Q000379:methods; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods
chemical_list: D012333:RNA, Messenger; D016601:RNA-Binding Proteins
keywords: RBP; RNA affinity pulldown; Ribonucleoprotein complexes; lncRNA