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.