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

Abstract

Long non-coding RNAs (LncRNAs) are non-protein coding transcripts longer than 200 nucleotides. Recent studies have revealed that nearly 80% of transcripts in human cells are lncRNA species. Based on their genomic location, most lncRNAs can be characterized as large intergenic non-coding RNAs, natural antisense transcripts, pseudogenes, and long intronic ncRNAs, as well as other divergent transcripts. However, despite mounting evidence 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) and 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 lncRNA interacting protein partners in a cellular context. We provide a detailed protocol for this assay with hands-on tips based on our own experience working in the lncRNA field.

Keywords: Long non-coding RNAs; Mass spectrometry; RNA–protein interaction.