MS2 tagging is a technique based upon the natural interaction of the MS2 bacteriophage coat protein with a stem-loop structure from the phage genome. Used for biochemical purification of RNA-protein complexes and partnered to GFP for detection of RNA in living cells. More recently, the technique has been used to monitor the appearance of RNA in living cells, at the site of transcription, or simply by observing the changes in RNA number in the cytoplasm. This has revealed that transcription of both prokaryotic and eukaryotic genes occurs in a discontinuous fashion (see Transcriptional bursting) with bursts of transcription separated by irregular intervals.
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The procedure
Start with single-stranded RNA and create a pattern of stem-loop structures by adding copies of MS2 RNA-binding sequences to a noncoding region. The MS2 protein needs to be fused with GFP and bonded to an mRNA, a complex that contains MS2’s RNA-binding sequence copies. Express the MS2-GFP fusion protein by transferring it to a cell with a plasmid (Robert Singer’s lab). Signal encodes within RNA and signal presences nuclear localization signal (NLS) within GFP-MS2 are two signals that introduce from EGFP-MS2-RNA complexes.
MS2 biotin-tagged RNA affinity purification (MS2-BioTRAP) is one method of identifying protein-RNA interactions. This method in vivo has to be expressed either side the RNA that tagged with MS2 and the MS2 protein tag, then the affinity interaction will use to help the process.
Advantages and Disadvantages
Disadvantages:
One caveat of MS2 tagging is that many copies of the MS2 stem-loop inside the RNA need to be added to produce enough signal for visibility and tracking one RNA molecule in the nucleus. When tracking more than one RNA sequence in the nucleus of cultured cells, more than one target sequence is needed. This could be affected by the MS2 protein, which has a classical basic nuclear localization signal (NLS), meaning it could affect the location of the RNA complex, and the nucleus would have most of the GFP-MS2 (Robert Singer’s lab),. The accumulation of GFP-MS2 in the nucleus will result in strong nuclear fluorescence signals, which will delay or prevent analysis of RNA nuclear localization because it will hinder the analysis of splicing, RNA editing, nuclear export of RNA, and RNA translation. Moreover, because of tag addition, the RNA secondary structure may introduce an artifact.
Additionally, the expression levels of small noncoding RNAS (sRNAs) and its regulatory properties will influence by MS2 tag. Also, using MS2 as an affinity tag to purify a protein in E. coli bacteria, scientists expressed the MS2-MBP, which is an MS2 coat protein carrying mutations fused with maltose-binding proteins. The mutations prevented oligomerization.
Advantages:
The MS2-BioTRAP method is fast, flexible, and easy to set up; it scales well and allows the study of the physiological conditions of protein-RNA interactions. Another advantage was demonstrated when scientists discovered another advantage when they successfully used an MS2 coat protein to isolate a variety of ribonucleoprotein particles (RNPs), as the MS2 tag is effective for small molecules.