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The Stem-Loop Luciferase Assay for Polyadenylation (SLAP) Method for Determining CstF-64-Dependent Polyadenylation Activity

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Polyadenylation

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1125))

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.

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Author information

Authors and Affiliations

  1. Department of Biology, University of the Cumberlands, Williamsburg, KY, USA

    J. Andrew Hockert

  2. Department of Cell Biology and Biochemistry, Texas Tech University Health Sciences Center, Lubbock, TX, USA

    Clinton C. MacDonald

Authors
  1. J. Andrew Hockert
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  2. Clinton C. MacDonald
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Editor information

Editors and Affiliations

  1. MRC Mitochondrial Biology Unit, Mitochondrial Genetics Group, Cambridge, United Kingdom

    Joanna Rorbach

  2. Mitochondrial Research Group, Newcastle University The Medical School, Newcastle upon Tyne, United Kingdom

    Agnieszka J. Bobrowicz

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Hockert, J.A., MacDonald, C.C. (2014). The Stem-Loop Luciferase Assay for Polyadenylation (SLAP) Method for Determining CstF-64-Dependent Polyadenylation Activity. 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_9

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  • DOI: https://doi.org/10.1007/978-1-62703-971-0_9

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-970-3

  • Online ISBN: 978-1-62703-971-0

  • eBook Packages: Springer Protocols

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