Oligoribonuclease functions as a diribonucleotidase to bypass a key bottleneck in RNA degradation

Degradation of RNA polymers is a multistep process catalyzed by specific subsets of RNases. In all cases, degradation is completed by exoribonucleases that recycle RNA fragments into nucleotide monophosphate. In γ-proteobacteria, a group of up to eight 3’-5’ exoribonucleases have been implicated in RNA degradation. Oligoribonuclease (Orn) is unique among them as its activity is required for clearing short RNA fragments, a function important for cellular fitness. However, the mechanistic basis for this substrate selectivity remained unclear. Here we show that Orn’s activity as a general exoribonuclease has been vastly overestimated by demonstrating that the enzyme exhibits a much narrower substrate preference for diribonucleotides. Co-crystal structures of Orn with substrates reveal an active site optimized for diribonucleotides that does not accommodate longer substrates. While other cellular RNases process oligoribonucleotides down to diribonucleotide entities, our functional studies demonstrate that Orn is the one and only diribonucleotidase that completes the final stage of the RNA degradation pathway. Together, these results indicate that Orn is a dedicated diribonucleotidase that clears the diribonucleotide pool that otherwise affects cellular physiology and viability.