![]() ![]() It is currently unclear whether such processing generates the active sRNAs, as is the case with eukaryotic microRNAs ( Kim, 2005). However, some primary sRNAs such as ArcZ and RprA are converted into shorter stable species that retain the seed region for target mRNA recognition ( Mandin and Gottesman, 2010, Papenfort et al., 2009, Papenfort et al., 2015). ![]() Many of the bacterial sRNAs characterized to date are transcribed from non-coding intergenic regions and operate as full-length, primary transcripts capped with a 5′ triphosphate (5′ PPP). A full understanding of these sRNA-mediated networks requires knowledge of how their RNA constituents are synthesized and turned over. ![]() ![]() These sRNAs generally act as multi-target repressors and activators through seed pairing interactions with the 5′ untranslated region (UTR) of mRNAs ( Desnoyers et al., 2013, Feng et al., 2015, Papenfort and Vanderpool, 2015). Initially defined as a class in non-pathogenic Escherichia coli ( Zhang et al., 2003), Hfq-dependent sRNAs have been globally mapped in numerous important human pathogens ( Barquist and Vogel, 2015, Holmqvist et al., 2016, Koo et al., 2011, Melamed et al., 2016, Tree et al., 2014). Small, non-coding RNAs (sRNAs) that associate with the RNA chaperone Hfq constitute the largest class of post-transcriptional regulators in Gram-negative bacteria ( De Lay et al., 2013, Storz et al., 2011, Vogel and Luisi, 2011, Wagner and Romby, 2015). Our findings reveal a general maturation mechanism for a major class of post-transcriptional regulators. In vivo, the processing is required for target regulation. Recapitulating this process in vitro, Hfq guides RNase E cleavage of a representative small-RNA precursor for interaction with a mRNA target. 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. 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. 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. Understanding RNA processing and turnover requires knowledge of cleavages by major endoribonucleases within a living cell. ![]()
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