Decay-initiating endoribonucleolytic cleavage by RNase Y is kept under tight control via sequence preference and sub-cellular localisation. Staphylococcus aureus

Bacteria depend on efficient RNA turnover to rapidly alter gene expression, essentially for responding to changing conditions. Nevertheless, remarkably few details are known about the rate-limiting steps in targeting and decay of RNA. The membrane-anchored endoribonuclease RNase Y is a virulence factor in Gram- positive pathogens. We have obtained a global picture of RNase Y sequence specificity using RNA-seq and the novel transcriptome-wide EMOTE method. Ninety- nine endoribonucleolytic sites produced in vivo were precisely mapped, notably inside six out of seven genes whose half-lives increase the most in an RNase Y deletion mutant, and additionally to three separate transcripts encoding degradation ribonucleases, including RNase Y itself, suggesting a regulatory network. We show that RNase Y is required to initiate the major degradation pathway of a defined sub-set of transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines. Overall design: Rnase Y activity in S. aureus is analysed on a genome wide scale under two perspectives: a RNA decay timecourse with mRNA-seq; and exact position of cleavage with an EMOTE assay (Exact Mapping Of Trancripts Ends)