Protocol

mRNA Processing and Metabolism

Volume 257 of the series Methods in Molecular Biology™ pp 193-211

Analysis of RNA Exonucleolytic Activities in Cellular Extracts

  • Devi MukherjeeAffiliated withDepartment of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School
  • , David T. FritzAffiliated withDepartment of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School
  • , Walter J. KilpatrickAffiliated withDepartment of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School
  • , Min GaoAffiliated withDepartment of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical School
  • , Jeffrey WiluszAffiliated withDepartment of Microbiology and Molecular Genetics, UMDNJ–New Jersey Medical SchoolDepartment of Microbiology, Immunology and Pathology, Colorado State University

* Final gross prices may vary according to local VAT.

Get Access

Abstract

Three types of exonucleases contribute to the turnover of messenger RNAs in eukaryotic cells: (1) general 3′-to-5′ exonucleases, (2) poly(A)-specific 3′-to-5′ exonucleases, and (3) 5′-to-3′ exonucleases. All three of these activities can be detected in cytoplasmic extracts from a variety of eukaryotic cells. In this chapter, we describe the preparation and use of HeLa cytoplasmic S100 extracts to study these three distinct exonuclease activities. Also included is an immunodepletion protocol that can be used to identify the enzyme responsible for a given activity. These protocols can be easily expanded to the study of trans-acting factors, cis-acting RNA sequence elements, and the interplay of components involved in RNA turnover in various mammalian cell types.

Key Words

Deadenylation mRNA turnover in vitro assay cytoplasmic extracts exonuclease exosome phosphothioates poly(A) mRNA stability