title: In vitro analysis of RNA degradation catalyzed by deadenylase enzymes.
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.
authors.affiliation: Genetics Training Program, Department of Biological Chemistry, University of Michigan Medical School, 1150 West Medical Center Dr., Room 5301 MSRB3, SPC 5606, Ann Arbor, MI, 48109, USA.
authors.forename: Joel
authors.initials: J
authors.lastname: Hrit
authors.forename: Nathan
authors.initials: N
authors.lastname: Raynard
authors.forename: Jamie
authors.initials: J
authors.lastname: Van Etten
authors.forename: Kamya
authors.initials: K
authors.lastname: Sankar
authors.forename: Adam
authors.initials: A
authors.lastname: Petterson
authors.forename: Aaron C
authors.initials: AC
authors.lastname: Goldstrohm
mesh_terms: D005095:Exoribonucleases / Q000378:metabolism; D011061:Poly A / Q000737:chemistry* / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry* / Q000235:genetics
chemical_list: D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D005095:Exoribonucleases