Purifi cation of Eukaryotic exoribonucleases following heterologous expression in bacteria and analysis of their biochemical properties by in vitro enzymatic assays

Abstract

Exoribonucleases—among the other RNases—play a crucial role in the regulation of different aspects of RNA metabolism in the eukaryotic cell. To fully understand the exact mechanism of activity exhibited by such enzymes, it is crucial to determine their detailed biochemical properties, notably their substrate specifi city and optimal conditions for enzymatic action. One of the most signifi cant features of exoribonucleases is the direction of degradation of RNA substrates, which can proceed either from 5′-end to 3′-end or in the opposite way. Here, we present methods allowing the effi cient production and purifi cation of eukaryotic exoribonucleases, the preparation and labeling of various RNA substrates, and the biochemical characterization of exonucleolytic activity. We also explain how the exonucleolytic activity may be distinguished from that of endonucleases.