RNA Degradation Assay Using RNA Exosome Complexes, Affinity-purified 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 RNA exosome-associated EXOSC10 protein is a distributive, 3'-5' exoribonuclease. The following protocol describes an approach to monitor the ribonucleolytic activity of affinity-purified EXOSC10-containing RNA exosomes, originating from HEK-293 cells, as reported in (Domanski et al., 2016) and further detailed in the companion bio-protocol to this one (Domanski and LaCava, 2017).

Keywords: Affinity capture; EXOSC10; RNA degradation; RNA exosome; Ribonuclease.

Figure 1.. Representative result: RNA degradation assay using affinity-purified human RNA exosome complexes.

Schematic diagram of a urea-polyacrylamide gel separation of the reaction products of an exosome RNase assay as described in this protocol, consistent with a distributive activity pattern and the original data. B. The original gel, reproduced from ( Domanski et al., 2016 ): an ExoI preparation was incubated either with a generic or blocked substrate and the indicated time points were collected.