title: Affinity purification of long noncoding RNA-protein complexes from formaldehyde cross-linked mammalian cells.
abstract: Long noncoding RNAs (lncRNAs) are a class of recently identified untranslated RNA molecules that have been shown to function in diverse cellular processes. The purification and analysis of lncRNA-protein (lncRNP) complexes is critical toward understanding the normal physiological function of these molecules. Here, we describe the purification of lncRNP complexes from human cells using a FLAG-tagged MS2-phage coat protein (MS2 CP) that binds in sequence-specific fashion to MS2-phage coat protein-binding sites (MS2bs) with high affinity. In these experiments, a FLAG-tagged MS2 CP is transiently co-expressed with a version of the lncRNA into which 12 copies of the MS2bs have been inserted near its 3'-end. The lncRNA-FLAG-tagged MS2 CP complex is then isolated using an anti-FLAG antibody, allowing for characterization of associated cellular proteins and RNAs.
authors.affiliation: Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, 601 Elmwood Ave., Rochester, NY, 14642, USA.
authors.forename: Chenguang
authors.initials: C
authors.lastname: Gong
authors.forename: Lynne E
authors.initials: LE
authors.lastname: Maquat
mesh_terms: D000818:Animals; D001665:Binding Sites; D036022:Capsid Proteins / Q000235:genetics / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003432:Cross-Linking Reagents / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D017909:Levivirus / Q000737:chemistry; D008322:Mammals; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D036022:Capsid Proteins; D003432:Cross-Linking Reagents; D062085:RNA, Long Noncoding; D005557:Formaldehyde