Human genome sequencing studies have shown that only 2 % of the human genome consists of protein-coding sequences. They have also shown that up to 90 % of the human genome can be transcribed. The transcripts from nonprotein-coding regions include noncoding RNAs. Long noncoding RNAs (lncRNAs) are a novel class of noncoding RNAs. lncRNAs have been shown to have important functions in development, as well as differentiation, X-chromosome inactivation, genomic imprinting, and cellular processes including cell cycle and apoptosis. They have also been implicated in human diseases ranging from cancer and amyotrophic lateral sclerosis (ALS) to Alzheimer’s disease. Recent studies have shown that lncRNAs function through gene regulation in almost all steps of gene expression including chromatin remodeling, transcription, splicing, RNA decay, translation, enhancer function , and epigenetic regulation. Gene regulation by lncRNAs requires protein complexes associated with lncRNAs. Thus, identification of proteins associated with lncRNAs is critical for the understanding of molecular mechanisms and functions of lncRNAs. Immunoprecipitation is commonly used for the isolation of protein complexes associated with a protein of interest. However, this method is not available for lncRNAs because antibodies generally do not recognize RNAs.
Here, researchers from the Wistar Institute describe a method to isolate protein complexes associated with lncRNAs using fusion protein maltose-binding protein (MBP) fused to MS2-binding protein.
MS2-binding protein binds to RNA sequence MS2 . The MS2 sequence needs to be added to either the 5′ or 3′ end of the lncRNA of interest. Sometimes adding the MS2 sequence to the end of lncRNA may change the structure of lncRNA and cause the loss of its functions. Functions of lncRNA-MS2 need to be confirmed by assays to ensure that the fusion lncRNA-MS2 RNA has the same function as the wild-type lncRNA. MBP-MS2 pull-down assay consists of the following steps. The first step involves the expression of MBP fused to MS2-binding protein (MBP-MS2) and immobilization of fusion protein to amylose beads. This fusion protein is ~59 kDa with MBP N-terminal to MS2-binding protein which carries a double mutation (V75Q and A81G) that prevents oligomerization and increases its affinity for RNA. The second step consists of the construction of plasmids and their expression in mammalian cells. A plasmid with MS2 sequence alone without any noncoding RNA is used as a control. Finally, transduce the cell line of interest with MS2 control or lncRNA-MS2 plasmid and lyse the cells. Incubate cytoplasmic or nuclear lysate with MBP-MS2-bound amylose beads, wash, and use for further analysis. PAGE and mass spectroscopy are used to identify the bound proteins.