Biochemical studies of the mammalian exosome with intact cells.
A key component responsible for 3'- to 5'-RNA turnover in eukaryotic cells is the exosome, a multisubunit complex present in both the nucleus and cytoplasm of the cell. Here we describe several methods that can be applied to study the structure and function of the exosome in mammalian cell lines. The mammalian two-hybrid system has been successfully used to identify protein-protein interactions between exosome core components. Cell and glycerol gradient fractionation procedures are described that allow the identification and characterization of different exosome subsets. Finally, a protocol to study the function of the exosome in RNA turnover with RNA interference is presented.
Schilders
Geurt
G
Department of Biomolecular Chemistry, Nijmegen Center for Molecular Life Sciences, Institute for Molecules and Materials, Radboud University Nijmegen, Nijmegen, The Netherlands.
Pruijn
Ger J M
GJ
D000818:Animals; D005095:Exoribonucleases / Q000378:metabolism*; D055354:Exosomes / Q000378:metabolism*; D005990:Glycerol; D006801:Humans; D011485:Protein Binding; D034622:RNA Interference; D020798:Two-Hybrid System Techniques
D005095:Exoribonucleases; D005990:Glycerol