title: Analysis of RNA exonucleolytic activities in cellular extracts.
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 <I>trans-acting factors, <I>cis-acting RNA sequence elements, and the interplay of components involved in RNA turnover in various mammalian cell types.
authors.affiliation: Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, Newark, NJ, USA.
authors.forename: Devi
authors.initials: D
authors.lastname: Mukherjee
authors.forename: David T
authors.initials: DT
authors.lastname: Fritz
authors.forename: Walter J
authors.initials: WJ
authors.lastname: Kilpatrick
authors.forename: Min
authors.initials: M
authors.lastname: Gao
authors.forename: Jeffrey
authors.initials: J
authors.lastname: Wilusz
mesh_terms: D002457:Cell Extracts / Q000302:isolation & purification*; D003593:Cytoplasm; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D011061:Poly A / Q000378:metabolism*; D012313:RNA / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D009418:S100 Proteins; D014158:Transcription, Genetic
chemical_list: D002457:Cell Extracts; D012333:RNA, Messenger; D009418:S100 Proteins; D011061:Poly A; D012313:RNA; D005092:Exonucleases