Branislav Večerek

Both RNase E and RNase III control the stability of sodB mRNA upon translational inhibition by the small regulatory RNA RyhB

Previous work has demonstrated that iron-dependent variations in the steady-state concentration and translatability of sodB mRNA are modulated by the small regulatory RNA RyhB, the RNA chaperone Hfq and RNase E. In agreement with the proposed role of RNase E, we found that the decay of sodB mRNA is retarded upon inactivation of RNase E in vivo, and that the enzyme cleaves within the sodB 5′-untranslated region (5′-UTR) in vitro, thereby removing the 5′ stem–loop structure that facilitates Hfq and ribosome binding. Moreover, RNase E cleavage can also occur at a cryptic site that becomes available upon sodB 5′-UTR/RyhB base pairing. We show that while playing an important role in facilitating the interaction of RyhB with sodB mRNA, Hfq is not tightly retained by the RyhB–sodB mRNA complex and can be released from it through interaction with other RNAs added in trans. Unlike turnover of sodB mRNA, RyhB decay in vivo is mainly dependent on RNase III, and its cleavage by RNase III in vitro is facilitated upon base pairing with the sodB 5′-UTR. These data are discussed in terms of a model, which accounts for the observed roles of RNase E and RNase III in sodB mRNA turnover.

Control of Fur synthesis by the non‐coding RNA RyhB and iron‐responsive decoding

The Fe2+‐dependent Fur protein serves as a negative regulator of iron uptake in bacteria. As only metallo‐Fur acts as an autogeneous repressor, Fe2+scarcity would direct fur expression when continued supply is not obviously required. We show that in Escherichia coli post‐transcriptional regulatory mechanisms ensure that Fur synthesis remains steady in iron limitation. Our studies revealed that fur translation is coupled to that of an upstream open reading frame (uof), translation of which is downregulated by the non‐coding RNA (ncRNA) RyhB. As RyhB transcription is negatively controlled by metallo‐Fur, iron depletion creates a negative feedback loop. RyhB‐mediated regulation of uof‐fur provides the first example for indirect translational regulation by a trans‐encoded ncRNA. In addition, we present evidence for an iron‐responsive decoding mechanism of the uof‐fur entity. It could serve as a backup mechanism of the RyhB circuitry, and represents the first link between iron availability and synthesis of an iron‐containing protein.

Interaction of the RNA chaperone Hfq with mRNAs: direct and indirect roles of Hfq in iron metabolism of Escherichia coli

The Escherichia coli Sm‐like host factor I (Hfq) is thought to play direct and indirect roles in post‐transcriptional regulation by targeting small regulatory RNAs and mRNAs. In this study, we have used proteomics to identify new mRNA targets of Hfq. We have identified 11 candidate proteins, synthesis of which was differentially affected in a hfq– background. The effect of Hfq on some of the corresponding mRNAs including fur, gapA, metF, ppiB and sodB mRNA was assessed, using different in vitro and in vivo methods. This allowed us to distinguish between direct and indirect effects of Hfq in modulating the translational activities of these mRNAs. From the collection of mRNAs tested, only fur and sodB mRNA, encoding the master regulator of iron metabolism and the iron superoxide dismutase, respectively, were found to be regulated by Hfq. Fur is known to be a negative regulator of transcription of the small RNA RyhB. Mutations in the sodB leader and compensating mutations in RyhB revealed that RyhB in turn represses translation of sodB mRNA, explaining the previously reported positive control of sodB by Fur. These data assign a role to Hfq in regulation of iron uptake and in switching off of iron scavenger genes.

Structural insights into the dynamics and function of the C-terminus of the E. coli RNA chaperone Hfq

The hexameric Escherichia coli RNA chaperone Hfq (HfqEc) is involved in riboregulation of target mRNAs by small trans-encoded RNAs. Hfq proteins of different bacteria comprise an evolutionarily conserved core, whereas the C-terminus is variable in length. Although the structure of the conserved core has been elucidated for several Hfq proteins, no structural information has yet been obtained for the C-terminus. Using bioinformatics, nuclear magnetic resonance spectroscopy, synchrotron radiation circular dichroism (SRCD) spectroscopy and small angle X-ray scattering we provide for the first time insights into the conformation and dynamic properties of the C-terminal extension of HfqEc. These studies indicate that the C-termini are flexible and extend laterally away from the hexameric core, displaying in this way features typical of intrinsically disordered proteins that facilitate intermolecular interactions. We identified a minimal, intrinsically disordered region of the C-terminus supporting the interactions with longer RNA fragments. This minimal region together with rest of the C-terminal extension provides a flexible moiety capable of tethering long and structurally diverse RNA molecules. Furthermore, SRCD spectroscopy supported the hypothesis that RNA fragments exceeding a certain length interact with the C-termini of HfqEc.

Requirement of the CsdA DEAD-box helicase for low temperature riboregulation of rpoS mRNA.

The ribosome binding site of Escherichia coli rpoS mRNA, encoding the stationary sigma-factor RpoS, is sequestered by an inhibitory stem-loop structure (iss). Translational activation of rpoS mRNA at low temperature and during exponential growth includes Hfq-facilitated duplex formation between rpoS and the small regulatory RNA DsrA as well as a concomitant re-direction of RNAse III cleavage in the 5´-untranslated region of rpoS upon DsrA·rpoS annealing. In this way, DsrA-mediated regulation does not only activate rpoS translation by disrupting the inhibitory secondary structure but also stabilizes the rpoS transcript. Although minor structural changes by Hfq have been observed in rpoS mRNA, a prevailing question concerns unfolding of the iss in rpoS at low growth temperature. Here, we have identified the DEAD-box helicase CsdA as an ancillary factor required for low temperature activation of RpoS synthesis by DsrA. The lack of RpoS synthesis observed in the csdA mutant strain at low growth temperature could be attributed to a lack of duplex formation between rpoS and DsrA, showing that at low temperature the sole action of Hfq is not sufficient to permit DsrA·rpoS annealing. An interactome study has previously indicated an association between Hfq and CsdA. However, immunological assays did not reveal a physical interaction between Hfq and CsdA. These findings add to a model, wherein Hfq binds upstream of the rpoS iss and presents DsrA in a conformation receptive to annealing. Melting of the iss by CsdA may then permit DsrA·rpoS duplex formation, and consequently rpoS translation.

Translational activation of rpoS mRNA by the non-coding RNA DsrA and Hfq does not require ribosome binding.

At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind.

Structural flexibility of RNA as molecular basis for Hfq chaperone function

In enteric bacteria, many small regulatory RNAs (sRNAs) associate with the RNA chaperone host factor Q (Hfq) and often require the protein for regulation of target mRNAs. Previous studies suggested that the hexameric Escherichia coli Hfq (HfqEc) binds sRNAs on the proximal site, whereas the distal site has been implicated in Hfq–mRNA interactions. Employing a combination of small angle X-ray scattering, nuclear magnetic resonance and biochemical approaches, we report the structural analysis of a 1:1 complex of HfqEc with a 34-nt-long subsequence of a natural substrate sRNA, DsrA (DsrA34). This sRNA is involved in post-transcriptional regulation of the E. coli rpoS mRNA encoding the stationary phase sigma factor RpoS. The molecular envelopes of HfqEc in complex with DsrA34 revealed an overall asymmetric shape of the complex in solution with the protein maintaining its doughnut-like structure, whereas the extended DsrA34 is flexible and displays an ensemble of different spatial arrangements. These results are discussed in terms of a model, wherein the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq.

Structural analysis of full-length Hfq from Escherichia coli

The structure of full-length host factor Qβ (Hfq) from Escherichia coli obtained from a crystal belonging to space group P1, with unit-cell parameters a = 61.91, b = 62.15, c = 81.26 Å, α = 78.6, β = 86.2, γ = 59.9°, was solved by molecular replacement to a resolution of 2.85 Å and refined to R(work) and R(free) values of 20.7% and 25.0%, respectively. Hfq from E. coli has previously been crystallized and the structure has been solved for the N-terminal 72 amino acids, which cover ~65% of the full-length sequence. Here, the purification, crystallization and structural data of the full 102-amino-acid protein are presented. These data revealed that the presence of the C-terminus changes the crystal packing of E. coli Hfq. The crystal structure is discussed in the context of the recently published solution structure of Hfq from E. coli.

Impact of Hfq on the Bacillus subtilis Transcriptome

The RNA chaperone Hfq acts as a central player in post-transcriptional gene regulation in several Gram-negative Bacteria, whereas comparatively little is known about its role in Gram-positive Bacteria. Here, we studied the function of Hfq in Bacillus subtilis, and show that it confers a survival advantage. A comparative transcriptome analysis revealed mRNAs with a differential abundance that are governed by the ResD-ResE system required for aerobic and anaerobic respiration. Expression of resD was found to be up-regulated in the hfq− strain. Furthermore, several genes of the GerE and ComK regulons were de-regulated in the hfq− background. Surprisingly, only six out of >100 known and predicted small RNAs (sRNAs) showed altered abundance in the absence of Hfq. Moreover, Hfq positively affected the transcript abundance of genes encoding type I toxin-antitoxin systems. Taken the moderate effect on sRNA levels and mRNAs together, it seems rather unlikely that Hfq plays a central role in RNA transactions in Bacillus subtilis.

The RNA chaperone Hfq is required for virulence of Bordetella pertussis.

ABSTRACT Bordetella pertussis is a Gram-negative pathogen causing the human respiratory disease called pertussis or whooping cough. Here we examined the role of the RNA chaperone Hfq in B. pertussis virulence. Hfq mediates interactions between small regulatory RNAs and their mRNA targets and thus plays an important role in posttranscriptional regulation of many cellular processes in bacteria, including production of virulence factors. We characterized an hfq deletion mutant (Δhfq) of B. pertussis 18323 and show that the Δhfq strain produces decreased amounts of the adenylate cyclase toxin that plays a central role in B. pertussis virulence. Production of pertussis toxin and filamentous hemagglutinin was affected to a lesser extent. In vitro, the ability of the Δhfq strain to survive within macrophages was significantly reduced compared to that of the wild-type (wt) strain. The virulence of the Δhfq strain in the mouse respiratory model of infection was attenuated, with its capacity to colonize mouse lungs being strongly reduced and its 50% lethal dose value being increased by one order of magnitude over that of the wt strain. In mixed-infection experiments, the Δhfq strain was then clearly outcompeted by the wt strain. This requirement for Hfq suggests involvement of small noncoding RNA regulation in B. pertussis virulence.