User:DrarSoaad/sandbox

Critique an article (Strigolactone).

MS2 tagging

I found this article does not have enough information about this technique, so I am going to update and add some concepts as the procedure and add some Pros & Cons for this technique. Here a list some bibliography that I'm going to use for this article:  

·       Marchese, D., de Groot, N. S., Lorenzo Gotor, N., Livi, C. M., & Tartaglia, G. G. (2016). Advances in the characterization of RNA‐binding proteins. Wiley Interdisciplinary Reviews: RNA, 7(6), 793-810.

·       Said, N., Rieder, R., Hurwitz, R., Deckert, J., Urlaub, H., & Vogel, J. (2009). In vivo expression and purification of aptamer-tagged small RNA regulators. Nucleic Acids Research, gkp719.

·      Webb, S. (2012). Live and In Color, How to track RNA in living cells. Retrieved from http://www.the-scientist.com/?articles.view/articleNo/31890/title/Live-and-In-Color/

The procedure

Start with single-stranded RNA, and create a pattern of stem-loop structures by adding copies of the MS2 RNA-binding sequences to a noncoding region. The MS2 protein must be fused with GFP and bonded to an mRNA, a complex that contains the MS2’s RNA-binding sequence copies. The MS2-GFP fusion protein was expressed by transferring it to a cell with a plasmid Robert Singer’s lab. The signal encodes within RNA and the signal presences of the 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 in vivo method of identifying protein-RNA interactions. Both the RNA that tagged with MS2 and the MS2 protein tag were expressed, and then, the affinity interaction was used to help the process of identifying protein-RNA interactions. 

 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 to view and track 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), so 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 the analysis of RNA nuclear localization because it will hinder the analysis of splicing, RNA editing, the nuclear export of RNA, and RNA translation.

Moreover, due to the addition of the tag, the RNA secondary structure may introduce an artifact. Additionally, the small noncoding RNA (sRNA) expression levels and regulatory properties will be influenced by MS2 tag. Also, by using MS2 as an affinity tag to purify a protein in E. coli bacteria, scientists expressed 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. Scientists also discovered that the MS2 tag is effective for small molecules when they successfully used an MS2 coat protein to isolate a variety of ribonucleoprotein particles (RNPs).