title: The stem-loop luciferase assay for polyadenylation (SLAP) method for determining CstF-64-dependent polyadenylation activity.
abstract: Polyadenylation is an essential cellular process in eukaryotic cells (Edmonds M and Abrams R, J Biol Chem 235, 1142-1149, 1960; Zhao J et al., Microbiol Mol Biol Rev 63, 405-445, 1999; Edmonds M, Progr Nucleic Acid Res Mol Biol 71, 285-389, 2002). For this reason, it has been difficult to examine the functions of specific polyadenylation proteins in vivo. Here, we describe a cell culture assay that allows structure-function experiments on CstF-64, a protein that binds to pre-mRNAs downstream of the cleavage site for accurate and efficient polyadenylation. We also demonstrate that the stem-loop luciferase assay for polyadenylation (SLAP) accurately reflects CstF-64-dependent polyadenylation. This assay could be easily adapted to the study of other important RNA-binding proteins in polyadenylation.
authors.affiliation: Department of Biology, University of the Cumberlands, Williamsburg, KY, USA.
authors.forename: J Andrew
authors.initials: JA
authors.lastname: Hockert
authors.forename: Clinton C
authors.initials: CC
authors.lastname: Macdonald
mesh_terms: D001681:Biological Assay / Q000379:methods*; D006367:HeLa Cells; D006801:Humans; D026723:Polyadenylation / Q000502:physiology*; D011062:Polynucleotide Adenylyltransferase / Q000378:metabolism; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*
chemical_list: D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011062:Polynucleotide Adenylyltransferase