LncRNA Pulldown Combined with Mass Spectrometry to Identify the Novel LncRNA-Associated Proteins

Abstract

Long noncoding RNAs (LncRNAs) are nonprotein-coding transcripts longer than 200 nucleotides in length. The recent studies have revealed that at least nearly 80 % transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic noncoding RNAs, natural antisense transcripts, pseudogenes, long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidences suggesting that many lncRNAs are likely to be functional, only a small proportion has been demonstrated to be biologically and physiologically relevant due to their lower expression levels and current technique limitations. Thus, there is a greater need to design and develop new assays to investigate the real function of lncRNAs in depth in various systems. Indeed, several methods such as genome-wide chromatin immunoprecipitation-sequencing (ChIP-seq), RNA immunoprecipitation followed by sequencing (RIP-seq) have been developed to examine the genome localization of lncRNAs and their interacting proteins in cells. Here we describe an open-ended method, LncRNA pulldown assay, which has been frequently used to identify its interacting protein partners in the cellular context. Here we provide a detailed protocol for this assay with hands-on tips based on our own experience in working in the lncRNA fields.

Keywords: Long noncoding RNAs; Mass spectrometry; RNA-protein interaction.