Abstract
In this chapter, we describe a method for purification and analysis of the enzymatic activity of deadenylase enzymes. Nearly all eukaryotic messenger RNAs are modified at the 3′ end by the addition of an adenosine polymer: the poly-adenosine tail. The poly(A) tail plays a central role in protein expression and mRNA fate. The poly(A) tail promotes translation of the mRNA. Shortening of the poly(A) tail, referred to as deadenylation, reduces protein synthesis and initiates destruction of the mRNA. A specialized class of exoribonucleases, called deadenylase enzymes, carries out this process. Deadenylases are found throughout eukarya, but their functions remain largely unexplored. We present a detailed protocol to analyze deadenylase activity in vitro. First, recombinant deadenylase enzyme is over-expressed and purified from bacteria. Next, labeled RNA substrate is prepared. Deadenylation reactions are performed, and reaction products are analyzed by denaturing gel electrophoresis. Reaction rates are then determined quantitatively. Crucial controls and experimental parameters are described along with practical tips that promote success.
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Acknowledgements
We thank Nathan Blewett, Chase Weidmann, Dr. Trista Schagat, and Dr. Brad Hook for help in developing these methods. Nathan Raynard is supported by the Michigan Predoctoral Training Program in Genetics through NIH National Research Service Award 5T32GM007544-33. Aaron Goldstrohm was supported by a Research Scholar Grant RSG-13-080-01-RMC from the American Cancer Society. This work was also supported by a grant from the Edward Mallinckrodt Jr. Foundation.
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Hrit, J., Raynard, N., Van Etten, J., Sankar, K., Petterson, A., Goldstrohm, A.C. (2014). In Vitro Analysis of RNA Degradation Catalyzed by Deadenylase Enzymes. In: Rorbach, J., Bobrowicz, A. (eds) Polyadenylation. Methods in Molecular Biology, vol 1125. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-971-0_26
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DOI: https://doi.org/10.1007/978-1-62703-971-0_26
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