Affinity Purification of the RNA Degradation Complex, the Exosome, from HEK-293 Cells

Abstract

The RNA exosome complex plays a central role in RNA processing and regulated turnover. Present both in cytoplasm and nucleus, the exosome functions through associations with ribonucleases and various adapter proteins (reviewed in [Kilchert et al., 2016]). The following protocol describes an approach to purify RNA exosome complexes from HEK-293 cells, making use of inducible ectopic expression, affinity capture, and rate-zonal centrifugation. The obtained RNA exosomes have been used successfully for proteomic, structural, and enzymatic studies (Domanski et al., 2016).

Keywords: Affinity capture; Cryomilling; EXOSC10; HEK-293 suspension culture; RNA exosome; Rate-zonal centrifugation.

Figure 1.. Representative results: RNA exosomes purified from HEK-293 cells expressing EXOSC10-3xFLAG.

A. Schematic diagram of a stained SDS-polyacrylamide gel demonstrating protein bands consistent with the separation of EXOSC10-3xFLAG purified RNA exosomes obtained after sedimentation within a 10-40% v/v glycerol gradient. B. The original gel, reproduced from Domanski et al., 2016 . Separated proteins were visualized by silver staining. The arrow indicates the peak fraction.