Biochemical analysis of long non-coding RNA-containing ribonucleoprotein complexes

Long non-coding RNAs (lncRNAs), once relegated to junk products of the genome, are becoming better appreciated for the myriad functions they play in cellular processes. It is clear that for most of the cases studied, lncRNAs carry out their functions at least in part through interactions with proteins. Here we present two complementary biochemical methods for the analysis of lncRNA-containing ribonucleoprotein complexes, hereafter referred to as RNPs. The first strategy offers users the ability to purify RNPs based on a protein component and to analyze the spectrum of lncRNAs, other proteins, and, if present, other types of RNAs that are bound to it. The second makes use of a bacteriophage MS2 binding-site affinity-handle grafted onto an lncRNA of interest to investigate the proteins and RNAs that co-purify with the tagged RNA.

► Immunoprecipitation of lncRNA-containing protein complexes via either a protein component or an lncRNA component.
► Insertion of MS2 coat protein-binding sites into lncRNAs to provide an RNA-based affinity-handle.
► Analysis of lncRNAs by RT coupled to either semi-quantitative or quantitative PCR.