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Revision as of 00:35, 18 October 2018
Poly(A)-Tailing
Poly(A)-tails are a series of adenosine (A) nucleotides that are assembled in a string at the 3’ end of the mRNA, like a tail. Poly(A)-tails are important in the cells for both stabilising and signaling, marking the mRNA as ready to be used for different purposes [1]. In eukaryotic cells, Poly(A)-tails are made naturally in the cell for most mRNA strands [2]. In prokaryotes however, the tails are shorter, more uncommon, and in many cases such as in E.coli, there are no tails on most mRNA at all [3].
Because poly(A)-tails are a long sequence of the same nucleotide, they make good targets for primers of genes with otherwise unknown sequences. In the next step, we will be using primers that will bind to poly(A)-tails and thus, we need to synthesize them onto our RNA samples.
Experiment
The purified RNA sample retrieved from previous mRNA purification step is dissolved in nuclease free water and ready to use. The key reagent is the poly(A) polymerase, an enzyme that attach the adenosine nucleotides onto the mRNA that is directly added into the sample.
As you might already know no work is done for free, and that is the case for the poly(A) polymerase. Thereby ATP is added, which is an energy molecule that activates the poly(A) polymerase. The mRNA is then analyzed with gel-electrophoresis to confirm that the poly(A) tail attachment was a success. It can be noted that we had some difficulties in acquiring good results initially from this step, which included degradation and apparent non-existing tailing of the samples. After several attempts however, and a complete change in Poly(A)-tailing reagents, the procedure started to work properly.
Result
A successful poly(A) tailed mRNA has a complete chain of adenosines connected to the 3’UTR of the mRNA strand. Initially, the adition of polyA has been very inefficient due to among other reasons low enzyme concentrations. Using double the recommended amount of enzyme, we managed to successfully attach of polyA tail to RNA. To be able to clearly illustrate the polyadenylation, we have added polyA tails to RNA ladder as can be seen in Figure 1 . The shift in size is especially well visible for the lowest 200 bp band.
Equal enzyme concentrations as those used in the RNA ladder polyadenylation were used to attach polyA tails to the isolated mRNA. The increase in polyA polymerase concentration has resulted in significantly higher cDNA yields, as decribed here
Figure 1. A successful poly(A) tail attachment onto RiboRuler High Range RNA Ladder (Thermo Fisher Scientific). The polyadnenylation is clearly visible on the bottom 200 bp band.
Buzzwords
Primers: A primer is a short sequence of DNA or RNA, that will work as a starting point for DNA synthesis. The DNA polymerase used to catalyze this process can only add new nucleotides to an already existing strand of DNA. The polymerase attaches to the primer and progressing the synthesis at the 3’end, while copying the opposite strand.
Poly(A) polymerase: Polyadenylate polymerase uses ATP to build the poly(A) tail, consisting of adenosine monophosphate. Adenosine is usually found in its triphosphate form, where the polymerase cleaving off pyrophosphate using monophosphate units to ad to the tail.
ATP: Adenosine triphosphate (ATP) is an organic chemical that is capable of providing energy to e.g. chemical reactions. When used in metabolic processes, it is converted either to adenosine diphosphate (ADP) or to adenosine monophosphate (AMP).
Gel electrophoresis: Gel electrophoresis is a analysis and separation method of macromolecules, such as DNA and RNA, and their fragments based on charge and size. When the electric field is applied the negatively charged molecules will move through a matrix of agarose. Shorter molecules migrates farther, due to the easier mobility through the pores in the matrix.
References
[1] Wu X, Brewer G. 2012. The regulation of mRNA stability in Mammalian Cells: 2.0. Gene 500(1): 10-21.
[2] Hunt AG, Xu R, Addepalli B, Rao S, Forbe KP, Meeks LR, Xing D, Mo M, Zhao H, Bandyopadhyay A, Dampanaboina L, Marion A, Von Lanken C, Quinn Li Q. Arabidopsis mRNA polyadenylation machinery: comprehensive analysis of protein-protein interaction and gene expression profiling. 2008. BMC Genomics 9:220. doi: 10.1186/1471-2164-9.220
[3] Sarkar N. 1997. Polyadenylation of mRNA in prokaryotes. Annual Review of Biochemistry. 66(1):173-97