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 structurefunction 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.