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 specificity and optimal conditions for enzymatic action. One of the most significant 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 efficient production and purification 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.
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This work was supported by the National Science Centre within the grant allocated to RT on the basis of the decision number DEC-2011/01/D/NZ1/03510 and through a grant awarded to RT by the National Centre for Research and Development within the LIDER program (LIDER/35/46/L-3/11/NCBR/2012). RT was the recipient of a scholarship for outstanding young scientists from the Polish Ministry of Science and Higher Education.
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