Stem-loops direct precise processing of 3′ UTR-derived small RNA MicL

Increasing numbers of 3′UTR-derived small, regulatory RNAs (sRNAs) are being discovered in bacteria, most generated by cleavage from longer transcripts. The enzyme required for these cleavages has been reported to be RNase E, the major endoribonuclease in enterica bacteria. Previous studies investigating RNase E have come to a range of different conclusions regarding the determinants for RNase E processing. To better understand the sequence and structure determinants for the precise processing of a 3′ UTR-derived sRNA, we examined the cleavage of multiple mutant and chimeric derivatives of the 3′ UTR-derived MicL sRNA in vivo and in vitro. Our results revealed that tandem stem–loops 3′ to the cleavage site define optimal, correctly-positioned cleavage of MicL and probably other sRNAs. Moreover, our assays of MicL, ArcZ and CpxQ showed that sRNAs exhibit differential sensitivity to RNase E, likely a consequence of a hierarchy of sRNA features recognized by the endonuclease.