The exosome is the main cellular machinery responsible for degrading RNA molecules in the 3′-to-5′ direction and is conserved from archaea and bacteria to eukaryotes. The core consists of a nine-subunit barrel-like structure called Exo-9, which is catalytically active in archaea and bacteria; in eukaryotes, by contrast, the RNase activity of exosomes resides in an additional subunit, Rrp44. Conti and colleagues reconstituted and crystallized a Saccharomyces cerevisiae exosome containing Exo-9 and Rrp44, bound to a C-terminal region of another nuclease, Rrp6, that stabilized the complex, and an RNA duplex substrate with a 3′ overhang. The structure reveals the canonical overall architecture of Exo-9, with the C-terminal region of Rrp6 wrapped around the exosome. Rrp44 adopts a closed conformation, with the exoribonuclease region obstructing the exit of the central channel. The first base pair of the RNA duplex is melted just before entering the Exo-9 channel by structural features of the cap proteins Rrp4 and Rrp40—reminiscent of the way that polypeptides are unfolded when entering the proteasome. RNA is then threaded, in a single-stranded conformation, through the narrow entrance pore by sequence-unspecific base-stacking interactions. The channel widens and then narrows again roughly halfway through the barrel-shaped channel and veers sideways, as previously observed in the archaeal exosome. But whereas in the archaeal exosome this sideway channel leads to the phosphorolytic active site of Rrp41, in the yeast exosome the channel ends in the exoribonuclease region of Rrp44, capturing the RNA 3′ end as it exits the side of Exo-9. Thus, although the enzymatic mechanisms differ, the substrate-channelling mechanisms of exosome complexes are conserved from archaea and bacteria to eukaryotes. (Nature 495, 70–75, 2013)