title: Single-molecule approach to immunoprecipitated protein complexes: insights into miRNA uridylation.
abstract: Single-molecule techniques have been used for only a subset of biological problems because of difficulties in studying proteins that require cofactors or post-translational modifications. Here, we present a new method integrating single-molecule fluorescence microscopy and immunopurification to study protein complexes. We used this method to investigate Lin28-mediated microRNA uridylation by TUT4 (terminal uridylyl transferase 4, polyU polymerase), which regulates let-7 microRNA biogenesis. Our real-time analysis of the uridylation by the TUT4 immunoprecipitates suggests that Lin28 functions as a processivity factor of TUT4. Our new technique, SIMPlex (single-molecule approach to immunoprecipitated protein complexes), provides a universal tool to analyse complex proteins at the single-molecule level.
authors.affiliation: School of Biological Sciences and Creative Research Center, Seoul National University, Seoul 151-742, Korea; Kavli Institute of NanoScience and Department of BioNanoScience, Delft University of Technology, Lorentzweg 1, Delft 2628 CJ, The Netherlands.
authors.forename: Kyu-Hyeon
authors.initials: KH
authors.lastname: Yeom
authors.forename: Inha
authors.initials: I
authors.lastname: Heo
authors.forename: Jinwoo
authors.initials: J
authors.lastname: Lee
authors.forename: Sungchul
authors.initials: S
authors.lastname: Hohng
authors.forename: V Narry
authors.initials: VN
authors.lastname: Kim
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
mesh_terms: D001681:Biological Assay; D004268:DNA-Binding Proteins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods*; D035683:MicroRNAs / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism*; D011485:Protein Binding; D012316:RNA Nucleotidyltransferases / Q000378:metabolism*; D016601:RNA-Binding Proteins
chemical_list: D004268:DNA-Binding Proteins; C516326:LIN28B protein, human; D035683:MicroRNAs; D046912:Multiprotein Complexes; D016601:RNA-Binding Proteins; C487931:mirnlet7 microRNA, human; D012316:RNA Nucleotidyltransferases; C031687:UTP-RNA uridylyltransferase