Reconstitution of the Schizosaccharomyces pombe RNA Exosome

Abstract

In this chapter, we describe methods to clone, express, purify, and reconstitute active S. pombe RNA exosomes. Reconstitution procedures are similar to methods that have been successful for the human and budding yeast exosome systems using protein subunits purified from the recombinant host E. coli. By applying these strategies, we can successfully reconstitute the S. pombe noncatalytic exosome core as well as complexes that contain the exoribonucleases Dis3 and Rrp6, cofactors Cti1 (equivalent to budding yeast Rrp47) and Mpp6 as well as the RNA helicase Mtr4.