Abstract
SummaryUnderstanding 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.
Highlights
Small, non-coding RNAs 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)
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 50 triphosphate (50 PPP)
A Transcriptome-wide Map of RNase E Cleavage Sites In Vivo To globally map RNase E cleavage events in vivo, we profiled 50 ends of cellular transcripts by comparative RNA-seq before and 30 min after programmed inactivation of the enzyme using a temperature-sensitive rneTS mutant (Apirion and Lassar, 1978; Figueroa-Bossi et al, 2009). We refer to this approach, which builds upon work by Clarke and colleagues (Clarke et al, 2014) as transient inactivation of endoribonuclease followed by RNA-seq (TIER-Seq; see Figure 1A)
Summary
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). 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) These sRNAs generally act as multi-target repressors and activators through seed pairing interactions with the 50 untranslated region (UTR) of mRNAs (Desnoyers et al, 2013; Feng et al, 2015; Papenfort and Vanderpool, 2015). These findings suggest that sRNA processing is a prevalent event; both its functional relevance and the major responsible nuclease(s) remain to be established
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