title: Studying the composition of mRNPs in vitro using splicing-competent cell extracts.
abstract: The correct processing and faithful decoding of mRNAs during gene expression depends on the interaction with RNA-binding proteins (RBPs). The association of RBPs with pre-mRNAs starts during transcription by RNA polymerase II and undergoes constant remodeling during pre-mRNA processing and later steps of genes expression. Recently developed high throughput methods enabled to define RBP binding sites in vivo and to identify a large number of novel RBPs in eukaryotic cells. However, the detailed characterization of RBP-RNA interactions as well as the analysis of functional RNPs is greatly facilitated by well-defined in vitro systems. Here, we describe a versatile method to study the assembly and splicing-dependent remodeling of mRNPs in vitro. This method employs splicing-competent whole cell extracts (WCE) generated from transfected human embryonic kidney (HEK) 293 cells. FLAG-tagged proteins present in the WCE are incorporated into mRNPs in vitro and afterwards used to immunoprecipitate substrate RNAs. We outline the principles of purifying in vitro assembled mRNPs and provide detailed protocols for the preparation and use of whole cell extracts. Alternative purification strategies and RNA substrates are discussed.
authors.affiliation: University of Cologne, Institute for Genetics, Cologne, Germany.
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Steckelberg
authors.affiliation: University of Cologne, Institute for Genetics, Cologne, Germany. Electronic address: ngehring@uni-koeln.de.
authors.forename: Niels H
authors.initials: NH
authors.lastname: Gehring
mesh_terms: D001665:Binding Sites; D002457:Cell Extracts / Q000737:chemistry; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D008958:Models, Molecular; D009363:Neoplasm Proteins / Q000737:chemistry* / Q000235:genetics; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics; D011485:Protein Binding; D012319:RNA Polymerase II / Q000235:genetics / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: C459802:CASC3 protein, human; D002457:Cell Extracts; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C527299:SRRT protein, human; D012319:RNA Polymerase II; C487886:DDX39B protein, human; D053487:DEAD-box RNA Helicases
keywords: Exon junction complex; In vitro splicing; RNA binding protein; RNP assembly