Role of the 5′ end phosphorylation state for small RNA stability and target RNA regulation in bacteria

In enteric bacteria, several small RNAs (sRNAs) including MicC employ endoribonuclease RNase E to stimulate target RNA decay. A current model proposes that interaction of the sRNA 5′ monophosphate (5′P) with the N-terminal sensing pocket of RNase E allosterically activates cleavage of the base-paired target in the active site. In vivo evidence supporting this model is lacking. Here, we engineered a genetic tool allowing us to generate 5′ monophosphorylated sRNAs of choice in a controllable manner in the cell. Four sRNAs were tested and none performed better in target destabilization when 5′ monophosphorylated. MicC retains full activity even when RNase E is defective in 5′P sensing, whereas regulation is lost upon removal of its scaffolding domain. Interestingly, sRNAs MicC and RyhB that originate with a 5′ triphosphate group are dramatically destabilized when 5′ monophosphorylated, but stable when in 5′ triphosphorylated form. In contrast, the processing-derived sRNAs CpxQ and SroC, which carry 5′P groups naturally, are highly stable. Thus, the 5′ phosphorylation state determines stability of naturally triphosphorylated sRNAs, but plays no major role for target RNA destabilization in vivo. In contrast, the RNase E C-terminal half is crucial for MicC-mediated ompD decay, suggesting that interaction with Hfq is mandatory.