In vivo cleavage map illuminates the central role of RNase E in coding and noncoding RNA pathways. Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. We have employed TIER-Seq (transiently inactivating an endoribonuclease followed by RNA-Seq) to profile cleavage products of the essential endoribonuclease RNase E in Salmonella enterica. A dominating cleavage signature is the location of a uridine two nucleotides downstream in a single-stranded segment, which we rationalize structurally as a key recognition determinant that may favor RNase E catalysis. Our results suggest a prominent biogenesis pathway for bacterial regulatory small RNAs, whereby RNase E acts together with the RNA chaperone Hfq to liberate stable 3’-fragments from various precursor RNAs. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small RNA precursor for interaction with a mRNA target. In vivo, the processing is required for target regulation. Our findings reveal a general maturation mechanism for a major class of post- transcriptional regulators. Overall design: 8 total RNA samples from Salmonella WT and rne-3071(rne-TS) strains grown at 28°C with and without heat treatment (44°C) for 30min, in duplicate.