In Vivo Cleavage Map Illuminates the Central Role of RNase E in Coding and Non-coding RNA Pathways

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.

RNA target profiles direct the discovery of virulence functions for the cold-shock proteins CspC and CspE

“…Future work should address the relative importance of this newly described mechanism compared with previously described roles for CSPs as chaperones facilitating RNA melting (56) or promoting antitermination (15). This issue could be addressed by establishing in vivo UV crosslinking (4) for CspC/E to determine direct binding sites at a global level; these data then could be integrated with global profiling of transcript decay data obtained in rifampicin run-out experiments (57) and with available information on RNase E cleavage sites (27) to understand the degree to which this mechanism accounts for the large number of gene-expression changes in the absence of CspC and CspE.…”

“…In Salmonella, RNase E is the major ribonuclease responsible for turnover of mRNAs. Recent genome-wide mapping of RNase E cleavage sites identified several sites within the ecnB mRNA (27), suggesting transcript protection from cleavage as a potential mechanism of CspC and CspE action. Combining the ΔcspCE mutant with the rne701 allele expressing a truncated RNase E variant abolished the ΔcspCE-dependent reduction of ecnB mRNA levels, suggesting that RNase E is responsible for the decreased stability of ecnB mRNA in the absence of CspC and CspE (SI Appendix, Fig.…”

RNase Y-mediated regulation of the streptococcal pyrogenic exotoxin B

“…S7) suggesting that the sRNA is functional. Indeed, RNase processing represents a common mechanism to generate active sRNAs [34–36]. Alternatively, the entire 5′ UTR could act as a trans -acting RNA similar to the 5′ UTR of irvA mRNA of Streptococcus mutans , encoding a transcriptional repressor, that interacts with gbpC mRNA and prevents its cleavage by RNase J2 [37].…”

“…A similar sequence preference has been recently described for RNase E, the functional equivalent of RNase Y in Gram-negative bacteria. Global mapping of RNase E processing events in Salmonella enterica revealed the preference for a uridine (U), 2 nt downstream of the cleavage site [34]. Mutation of this nucleotide strongly reduced the processing of the mRNA targets by RNase E, both in vitro and in vivo [34].…”

“…Global mapping of RNase E processing events in Salmonella enterica revealed the preference for a uridine (U), 2 nt downstream of the cleavage site [34]. Mutation of this nucleotide strongly reduced the processing of the mRNA targets by RNase E, both in vitro and in vivo [34]. Since structural data showed that RNase E interacts with an RNA substrate, 2 nt downstream of the cleavage site, the authors proposed a model in which the U at this position induces a conformational change in the catalytic site, which favors the processing.…”