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.