Abstract
As a result of its importance in key RNA metabolic processes, the ribonucleolytic RNA exosome complex has been the focus of intense study for almost two decades. Research on exosome subunit assembly, cofactor and substrate interaction, enzymatic catalysis and structure have largely been conducted using complexes produced in the yeast Saccharomyces cerevisiae or in bacteria. Here, we examine different populations of endogenous exosomes from human embryonic kidney (HEK) 293 cells and test their enzymatic activity and structural integrity. We describe methods to prepare EXOSC10-containing, enzymatically active endogenous human exosomes at suitable yield and purity for in vitro biochemistry and negative stain transmission electron microscopy. This opens the door for assays designed to test the in vitro effects of putative cofactors on human exosome activity and will enable structural studies of preparations from endogenous sources.
Highlights
The eukaryotic RNA exosome is a multisubunit complex harboring both exo- and endonuclease activities derived from enzyme components situated upon a catalytically inactive structural core that bears similarities to bacterial PNPase
To produce endogenous human RNA exosomes at largescale, we conditioned normally adherent human embryonic kidney (HEK)-293 Flp-In T-REx cells expressing tetracycline inducible 3xFlag-tagged EXOSC10 to suspension growth, which yielded ∼10 g wet cell weight (WCW) per 400 mL of culture medium
We utilized protocols incorporating scaled-up versions of our previously described methods (Domanski et al 2012; Hakhverdyan et al 2015), in conjunction with glycerol density gradient rate zonal centrifugation, to produce distinct exosome populations differentiated by the absence (ExoI) or presence (ExoII) of the component DIS3 (Fig. 1C)
Summary
The eukaryotic RNA exosome is a multisubunit complex harboring both exo- and endonuclease activities derived from enzyme components situated upon a catalytically inactive structural core that bears similarities to bacterial PNPase (for review, see Wasmuth and Lima 2012b). The abundance of exosome complexes in human cells is not a limiting factor (estimated at ∼40,000 copies per cell in log-phase U2OS cell cultures [Beck et al 2011]), preparing large enough quantities of cells expressing, but not overexpressing, tagged exosome complexes is relatively expensive and time consuming. An additional hurdle to studying human exosomes has been the lack of effective procedures to obtain abundant and pure complexes from the typically smaller quantity of starting material obtained from human tissue culture
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