Hibernation-Promoting Factor Sequesters Staphylococcus aureus Ribosomes to Antagonize RNase R-Mediated Nucleolytic Degradation

Bacterial and eukaryotic hibernation factors prevent translation by physically blocking the decoding center of ribosomes, a phenomenon called ribosome hibernation that often occurs in response to nutrient deprivation. The human pathogen Staphylococcus aureus lacking the sole hibernation factor HPF undergoes massive ribosome degradation via an unknown pathway. Using genetic and biochemical approaches, we find that inactivating the 3′-to-5′ exonuclease RNase R suppresses ribosome degradation in the Δhpf mutant. In vitro cell-free degradation assays confirm that 30S and 70S ribosomes isolated from the Δhpf mutant are extremely susceptible to RNase R, in stark contrast to nucleolytic resistance of the HPF-bound 70S and 100S complexes isolated from the wild type. In the absence of HPF, specific S. aureus 16S rRNA helices are sensitive to nucleolytic cleavage. These RNase hot spots are distinct from that found in the Escherichia coli ribosomes. S. aureus RNase R is associated with ribosomes, but unlike the E. coli counterpart, it is not regulated by general stressors and acetylation. The results not only highlight key differences between the evolutionarily conserved RNase R homologs but also provide direct evidence that HPF preserves ribosome integrity beyond its role in translational avoidance, thereby poising the hibernating ribosomes for rapid resumption of translation. IMPORTANCE Ribosome hibernation is pivotal for the rapid recovery of translation after quiescence in both bacteria and eukaryotes. Ribosome hibernation factors sterically occlude the entry of mRNA and tRNA and are thought to primarily maintain ribosomes in a translation-repressive state, thereby providing a pool of readily recyclable 70S or 80S complexes upon dissociation of the hibernation factors. Ribosomes in Staphylococcus aureus cells lacking the sole hibernation factor HPF are extremely unstable. Here, we show that HPF binding inhibits ribosome degradation by the evolutionarily conserved exoribonuclease RNase R. The data not only uncover a direct protective role of HPF in ribosome stability but also reinforce the versatility of RNase R in RNA processing, decay, and ribosome quality control.