Structure of a bacterial ribonucleoprotein complex central to the control of cell envelope biogenesis

The biogenesis of the essential precursor of the bacterial cell envelope, glucosamine-6-phosphate (GlcN6P), is controlled through intricate post-transcription networks mediated by GlmZ, a small regulatory RNA (sRNA). GlmZ stimulates translation of the mRNA encoding GlcN6P synthetase in Escherichia coli, but when bound by the protein RapZ, it becomes inactivated through cleavage by the endoribonuclease RNase E. Here we report the cryoEM structure of the RapZ:GlmZ complex, revealing a complementary match of the protein tetrameric quaternary structure to an imperfect structural repeat in the RNA. The RNA is contacted mostly through a highly conserved domain of RapZ that shares deep evolutionary relationship with phosphofructokinase and suggests links between metabolism and riboregulation. We also present the structure of a pre-cleavage encounter intermediate formed between the binary RapZ:GlmZ complex and RNase E that reveals how GlmZ is presented and recognised for cleavage. The structures suggest how other encounter complexes might guide recognition and action of endoribonucleases on target transcripts, and how structured substrates in polycistronic precursors are recognised for processing.

RNase III-mediated processing of a trans-acting bacterial sRNA and its cis-encoded antagonist

Bacterial small RNAs (sRNAs) are important post-transcriptional regulators in stress responses and virulence. They can be derived from an expanding list of genomic contexts, such as processing from parental transcripts by RNase E. The role of RNase III in sRNA biogenesis is less well understood despite its well-known roles in rRNA processing, RNA decay, and cleavage of sRNA-mRNA duplexes. Here, we show that RNase III processes a pair of cis-encoded sRNAs (CJnc190 and CJnc180) of the foodborne pathogen Campylobacter jejuni. While CJnc180 processing by RNase III requires CJnc190, In contrast, RNase III processes CJnc190 independent of CJnc180 via cleavage of an intramolecular duplex. We also show that CJnc190 directly represses translation of the colonization factor PtmG by targeting a G-rich ribosome binding site, and uncover that CJnc180 is a cis-acting antagonist of CJnc190, indirectly affecting ptmG regulation. Our study highlights a role for RNase III in sRNA biogenesis and adds cis-encoded RNAs to the expanding diversity of transcripts that antagonize bacterial sRNAs.

Ubiquitous mRNA decay fragments in E. coli redefine the functional transcriptome

Bacterial mRNAs have short life cycles, in which transcription is rapidly followed by translation and degradation within seconds to minutes. The resulting diversity of mRNAs impacts their functionality but has remained unresolved. Here we quantitatively map the 3' status of cellular RNAs in Escherichia coli during steady-state growth and report a large fraction of molecules (median>60%) that are fragments of canonical full-length mRNAs. The majority of RNA fragments are decay intermediates following endonuclease cleavage by RNase E and yet-unknown nucleases, whereas nascent RNAs contribute to a smaller fraction. Despite the prevalence of decay intermediates in total RNA, they are underrepresented in the pool of ribosome-associated transcripts and can thus distort quantifications for the abundance of full-length, functional mRNAs. The large heterogeneity within mRNA molecules in vivo highlights the importance in discerning functional transcripts and provides a lens for studying the dynamic life cycle of mRNAs.

Maturation of UTR-Derived sRNAs Is Modulated during Adaptation to Different Growth Conditions

Small regulatory RNAs play a major role in bacterial gene regulation by binding their target mRNAs, which mostly influences the stability or translation of the target. Expression levels of sRNAs are often regulated by their own promoters, but recent reports have highlighted the presence and importance of sRNAs that are derived from mRNA 3′ untranslated regions (UTRs). In this study, we investigated the maturation of 5′ and 3′ UTR-derived sRNAs on a global scale in the facultative phototrophic alphaproteobacterium Rhodobacter sphaeroides. Including some already known UTR-derived sRNAs like UpsM or CcsR1-4, 14 sRNAs are predicted to be located in 5′ UTRs and 16 in 3′ UTRs. The involvement of different ribonucleases during maturation was predicted by a differential RNA 5′/3′ end analysis based on RNA next generation sequencing (NGS) data from the respective deletion strains. The results were validated in vivo and underline the importance of polynucleotide phosphorylase (PNPase) and ribonuclease E (RNase E) during processing and maturation. The abundances of some UTR-derived sRNAs changed when cultures were exposed to external stress conditions, such as oxidative stress and also during different growth phases. Promoter fusions revealed that this effect cannot be solely attributed to an altered transcription rate. Moreover, the RNase E dependent cleavage of several UTR-derived sRNAs varied significantly during the early stationary phase and under iron depletion conditions. We conclude that an alteration of ribonucleolytic processing influences the levels of UTR-derived sRNAs, and may thus indirectly affect their mRNA targets.

Escherichia coli ribosomal protein S1 enhances the kinetics of ribosome biogenesis and RNA decay

Escherichia coli ribosomal protein S1 is essential for translation initiation of mRNAs and for cellular viability. Two oligonucleotide binding (OB)-fold domains located in the C-terminus of S1 are dispensable for growth, but their deletion causes a cold-shock phenotype, loss of motility and deregulation of RNA mediated stress responses. Surprisingly, the expression of the small regulatory RNA RyhB and one of its repressed target mRNA, sodB, are enhanced in the mutant strain lacking the two OB domains. Using in vivo and in vitro approaches, we show that RyhB retains its capacity to repress translation of target mRNAs in the mutant strain but becomes deficient in triggering rapid turnover of those transcripts. In addition, the mutant is defective in of the final step of the RNase E-dependent maturation of the 16S rRNA. This work unveils an unexpected function of S1 in facilitating ribosome biogenesis and RyhB-dependent mRNA decay mediated by the RNA degradosome. Through its RNA chaperone activity, S1 participates to the coupling between ribosome biogenesis, translation, and RNA decay.

A pathogen-specific sRNA influences enterohemorrhagic Escherichia coli fitness and virulence in part by direct interaction with the transcript encoding the ethanolamine utilization regulatory factor EutR

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 relies on sRNAs to coordinate expression of metabolic and virulence factors to colonize the host. Here, we focus on the sRNA, named MavR (metabolism and virulence regulator), that is conserved among pathogenic Enterobacteriaceae. MavR is constitutively expressed under in vitro conditions that promote EHEC virulence gene expression. Using MS2-affinity purification coupled with RNA sequencing, the eutR transcript was identified as a putative target of MavR. EutR is a transcription factor that promotes expression of genes required for ethanolamine metabolism as well as virulence factors important for host colonization. MavR binds to the eutR coding sequence to protect the eutR transcript from RNase E-mediated degradation. Ultimately, MavR promotes EutR expression and in turn ethanolamine utilization and ethanolamine-dependent growth. RNAseq analyses revealed that MavR also affected expression of genes important for other metabolic pathways, motility, oxidative stress and attaching and effacing lesion formation, which contribute to EHEC colonization of the gastrointestinal tract. In support of the idea that MavR-dependent gene expression affects fitness during infection, deletion of mavR resulted in significant (∼10- to 100-fold) attenuation in colonization of the mammalian intestine. Altogether, these studies reveal an important, extensive, and robust phenotype for a bacterial sRNA in host-pathogen interactions.

Binding of the RNA Chaperone Hfq on Target mRNAs Promotes the Small RNA RyhB-Induced Degradation in Escherichia coli

Many RNA-RNA interactions depend on molecular chaperones to form and remain stable in living cells. A prime example is the RNA chaperone Hfq, which is a critical effector involved in regulatory interactions between small RNAs (sRNAs) and cognate target mRNAs in Enterobacteriaceae. While there is a great deal of in vitro biochemical evidence supporting the model that Hfq enhances rates or affinities of sRNA:mRNA interactions, there is little corroborating in vivo evidence. Here we used in vivo tools including reporter genes, co-purification assays, and super-resolution microscopy to analyze the role of Hfq in RyhB-mediated regulation, and we found that Hfq is often unnecessary for efficient RyhB:mRNA complex formation in vivo. Remarkably, our data suggest that a primary function of Hfq is to promote RyhB-induced cleavage of mRNA targets by RNase E. Moreover, our work indicates that Hfq plays a more limited role in dictating regulatory outcomes following sRNAs RybB and DsrA complex formation with specific target mRNAs. Our investigation helps evaluate the roles played by Hfq in some RNA-mediated regulation.

Polyribosome-Dependent Clustering of Membrane-Anchored RNA Degradosomes To Form Sites of mRNA Degradation in Escherichia coli

Here, we show that RNase E, RhlB, and PNPase act together as components of the multienzyme RNA degradosome in polyribosome-dependent clustering to form puncta on the inner cytoplasmic membrane. Our results support the hypothesis that RNA degradosome puncta are sites of mRNA degradation.

RNase III and RNase E Influence Posttranscriptional Regulatory Networks Involved in Virulence Factor Production, Metabolism, and Regulatory RNA Processing in Bordetella pertussis

Noncoding, regulatory RNAs in bacterial pathogens are critical components required for rapid changes in gene expression profiles. However, little is known about the role of regulatory RNAs in the growth and pathogenesis of Bordetella pertussis .

Tailoring the evolution of BL21(DE3) uncovers a key role for RNA stability in gene expression toxicity

Gene expression toxicity is an important biological phenomenon and a major bottleneck in biotechnology. Escherichia coli BL21(DE3) is the most popular choice for recombinant protein production, and various derivatives have been evolved or engineered to facilitate improved yield and tolerance to toxic genes. However, previous efforts to evolve BL21, such as the Walker strains C41 and C43, resulted only in decreased expression strength of the T7 system. This reveals little about the mechanisms at play and constitutes only marginal progress towards a generally higher producing cell factory. Here, we restrict the solution space for BL21(DE3) to evolve tolerance and isolate a mutant strain Evo21(DE3) with a truncation in the essential RNase E. This suggests that RNA stability plays a central role in gene expression toxicity. The evolved rne truncation is similar to a mutation previously engineered into the commercially available BL21Star(DE3), which challenges the existing assumption that this strain is unsuitable for expressing toxic proteins. We isolated another dominant mutation in a presumed substrate binding site of RNase E that improves protein production further when provided as an auxiliary plasmid. This makes it easy to improve other BL21 variants and points to RNases as prime targets for cell factory optimisation.