Analysis of RNA Exonucleolytic Activities in Cellular Extracts
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 WordsDeadenylation mRNA turnover in vitro assay cytoplasmic extracts exonuclease exosome phosphothioates poly(A) mRNA stability
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