RNA and proteins are interrelated biomolecules that can physically affect each other's life cycle and function. RNA binding proteins (RBPs) play a key role in a variety of biological processes including RNA transcription, processing, modification, translocation, translation and stabilization, etc. RNA can also influence protein expression, localization and interaction with other proteins. Aberrant RNA-protein interactions have been associated with cellular dysfunction and human diseases. Therefore, studying RNA-protein interactions is important for understanding many cell biological processes and disease pathogenesis.
In vitro RNA-based methods to study RNA-protein interactions are mainly performed by tagging the target RNA molecule with either biotin, fluorescent dye, or some neck-loop structured Tag (e.g. S1, D8, MS2, etc.) MS2-BioTRAP is a method developed by Tsai et al. based on MS2 and biotin affinity purification technology to capture in vivo RNA-protein complexes in vivo.
Creative Proteomics offers an RNA-Protein interaction analysis service based on MS2-BioTRAP technology. This service enables the identification of proteins that specifically bind to an RNA of interest, and using this method, protein factors that interact with specific RNAs during RNA transcription, processing, localization, translation and degradation-related processes can be analyzed.
The core principle of MS2-BioTRAP technology is the ability of MS2 phage-derived MCP protein (MS2 coat protein) to bind with high affinity to single-stranded loop regions (MCP binding sites, MBS) on specific RNA stem-loop structures. The method first requires the construction of a cell line that stably expresses the MCP-HB fusion protein, a histidine andbiotin tag containing two 6<His sequences, a tobacco etch virus (TEV) cleavage site, and an endogenous biotinylated signal sequence. The biotinylation signal sequence can be catalyzed by endogenous biotin transferase with biotin tags for subsequent affinity purification. In addition, an expression plasmid with a tandem repeat of the MBS stem-loop sequence at the end of the target RNA needs to be constructed and transfected into the above stable cell line. The tight binding between the MCP and MBS stem-loop sequences makes HB tag-based affinity purification possible. Only affinity purification by streptavidin is required to obtain proteins specifically bound to the target RNA. The resulting proteins are then subsequently analyzed using mass spectrometry and protein immunoblotting.
MS2 in vivo biotin tagged RNA affinity purification (MS2-BioTRAP) and SILAC-based mass spectrometry (Tsai et al., 2011).
Reflects the true binding of RNA and protein in vivo, avoiding non-specific binding in vitro.
The method can be applied to any RNA of interest and is simple and fast. Simply add the MBS stem-loop sequence to the target sequence, eliminating the need to design separate purification probes for each individual RNA.
Both the target RNA and MCP are overexpressed in the cell, thus causing the problem of high false positive rate. When a large amount of MCP binds to RNA it may affect processes such as RNA secondary structure formation, affinity of related proteins to RNA, and RNA degradation.
Creative Proteomics has accumulated a wealth of experience in RNA-protein interaction research. Our team of technical professionals also offers other in vivo identification techniques for RNA-protein interaction analysis, including ChIRP, CHART and RAP-MS, etc. Contact us to learn more.