title: Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation.
abstract: The stabilization, translation and degradation of RNA are regulated by interactions between trans-acting factors, such as microRNA and RNA-binding proteins (RBP). In order to investigate the relationships between these events and their significance, a method that detects the localization of these interactions within a single cell, as well as their variability across a cell population, is needed. To visualize and quantify RNA-protein interactions in situ, we developed a proximity ligation assay (PLA) that combined peptide-modified, multiply-labelled tetravalent RNA imaging probes (MTRIPs), targeted to sequences near RBP binding sites, with proximity ligation and rolling circle amplification (RCA). Using this method, we detected and quantified, with single-interaction sensitivity, the localization and frequency of interactions of the human respiratory syncytial virus (hRSV) nucleocapsid protein (N) with viral genomic RNA (gRNA). We also described the effects of actinomycin D (actD) on the interactions of HuR with β-actin mRNA and with poly(A)+ mRNA at both native and increased HuR expression levels.
authors.affiliation: Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, 313 Ferst Drive, UA Whitaker Bldg, Atlanta, GA 30332, USA.
authors.forename: Jeenah
authors.initials: J
authors.lastname: Jung
authors.forename: Aaron W
authors.initials: AW
authors.lastname: Lifland
authors.forename: Chiara
authors.initials: C
authors.lastname: Zurla
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Alonas
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Santangelo
mesh_terms: D002460:Cell Line; D003609:Dactinomycin / Q000494:pharmacology; D051959:ELAV Proteins / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D057054:Molecular Imaging / Q000379:methods*; D015335:Molecular Probes / Q000737:chemistry; D019590:Nucleocapsid Proteins / Q000378:metabolism; D009842:Oligopeptides; D010455:Peptides / Q000737:chemistry; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D051959:ELAV Proteins; D015335:Molecular Probes; D019590:Nucleocapsid Proteins; D009842:Oligopeptides; D010455:Peptides; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D003609:Dactinomycin; D012313:RNA; C085706:FLAG peptide