Coordination of sequential RNase activities in an ancient molecular machine

The evolutionary mechanisms by which molecular machines incorporate catalytic subunits and coordinate their functions are still poorly understood. Here, we study the molecular evolution of the RNA exosome, an essential RNA-processing machine built from a 9-subunit core (Exo9). Human and yeast Exo9 are catalytically inactive and serve as a recruiting hub for the peripheral RNase Rrp44. The core of modern exosomes descends from an active RNase in Archaea. Using ancestral sequence reconstruction, biochemical and structural characterization, we illuminate how Exo9 evolved from an enzyme to a regulatory hub. The ancestral Exo9 was an active, distributive RNase that already cooperated with Rrp44. Cryo-electron microscopy reveals how RNA-binding modulates the conformation of Exo9, thereby promoting allosteric recruitment of Rrp44. Exo9 begins substrate trimming before the RNA slips past its active site. The RNA can thereby be handed over to Rrp44 to be processed further, which rationalizes the coordination of consecutive RNase activities. We hypothesize that the same allosteric pathway still exists in the human exosome, implying that this mechanism persisted for over a billion years. Our work thus illuminates the evolutionary tinkering that produced a complex molecular machine and a central component of the eukaryotic cell.