Hfq: a bacterial Sm-like protein that mediates RNA-RNA interaction.

The bacterial Hfq protein modulates the stability or the translation of mRNAs and has recently been shown to interact with small regulatory RNAs in E. coli. Here we show that Hfq belongs to the large family of Sm and Sm-like proteins: it contains a conserved sequence motif, known as the Sm1 motif, forms a doughnut-shaped structure, and has RNA binding specificity very similar to the Sm proteins. Moreover, we provide evidence that Hfq strongly cooperates in intermolecular base pairing between the antisense regulator Spot 42 RNA and its target RNA. We speculate that Sm proteins in general cooperate in bimolecular RNA-RNA interaction and that protein-mediated complex formation permits small RNAs to interact with a broad range of target RNAs.     

The OxyS regulatory RNA represses rpoS translation and binds the Hfq (HF-I) protein.

The OxyS regulatory RNA integrates the adaptive response to hydrogen peroxide with other cellular stress responses and protects against DNA damage. Among the OxyS targets is the rpoS-encoded sigma(s) subunit of RNA polymerase. Sigma(s) is a central regulator of genes induced by osmotic stress, starvation and entry into stationary phase. We examined the mechanism whereby OxyS represses rpoS expression and found that the OxyS RNA inhibits translation of the rpoS message. This repression is dependent on the hfq-encoded RNA-binding protein (also denoted host factor I, HF-I). Co-immunoprecipitation and gel mobility shift experiments revealed that the OxyS RNA binds Hfq, suggesting that OxyS represses rpoS translation by altering Hfq activity.     

Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA

Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA.rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA.rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA.rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA.mRNA complex.     

Dynamic Refolding of OxyS sRNA by the Hfq RNA Chaperone

The Sm protein Hfq chaperones small non-coding RNAs (sRNAs) in bacteria, facilitating sRNA regulation of target mRNAs. Hfq acts in part by remodeling the sRNA and mRNA structures, yet the basis for this remodeling activity is not understood. To understand how Hfq remodels RNA, we used single-molecule Förster resonance energy transfer (smFRET) to monitor conformational changes in OxyS sRNA upon Hfq binding. The results show that E. coli Hfq first compacts OxyS, bringing its 5′ and 3 ends together. Next, Hfq destabilizes an internal stem-loop in OxyS, allowing the RNA to adopt a more open conformation that is stabilized by a conserved arginine on the rim of Hfq. The frequency of transitions between compact and open conformations depend on interactions with Hfqs flexible C-terminal domain (CTD), being more rapid when the CTD is deleted, and slower when OxyS is bound to Caulobacter crescentus Hfq, which has a shorter and more stable CTD than E. coli Hfq. We propose that the CTDs gate transitions between OxyS conformations that are stabilized by interaction with one or more arginines. These results suggest a general model for how basic residues and intrinsically disordered regions of RNA chaperones act together to refold RNA.     

Sm-like protein Hfq: location of the ATP-binding site and the effect of ATP on Hfq-- RNA complexes

Sm-like proteins are ubiquitous ring-shaped oligomers that exhibit a variety of nucleic acid-binding activities. They have been linked functionally to various cellular events involving RNA, and it is generally believed that their activity is exerted via the passive binding of nucleic acids. Our earlier studies of the Sm-like Escherichia coli protein Hfq provided the first evidence indicating that Hfq is an ATP-binding protein. Using a combination of biochemical and genetic techniques, we have now determined a plausible ATP-binding site in Hfq and tested Hfq's ATP-binding affinity and stoichiometry. The results of RNA footprinting and binding analyses suggest that ATP binding by the Hfq-RNA complex results in its significant destabilization. RNA footprinting indicates deprotection of Hfq-bound RNA tracts in the presence of ATP, suggestive of their release by the protein. The results reported herein broaden the scope of potential in vivo roles for Hfq and other Sm-like proteins.     

Sm-like proteins in Eubacteria: the crystal structure of the Hfq protein from Escherichia coli

The Hfq protein was discovered in Escherichia coli in the early seventies as a host factor for the Qbeta phage RNA replication. During the last decade, it was shown to be involved in many RNA processing events and remote sequence homology indicated a link to spliceosomal Sm proteins. We report the crystal structure of the E.coli Hfq protein showing that its monomer displays a characteristic Sm-fold and forms a homo-hexamer, in agreement with former biochemical data. Overall, the structure of the E.coli Hfq ring is similar to the one recently described for Staphylococcus aureus. This confirms that bacteria contain a hexameric Sm-like protein which is likely to be an ancient and less specialized form characterized by a relaxed RNA binding specificity. In addition, we identified an Hfq ortholog in the archaeon Methanococcus jannaschii which lacks a classical Sm/Lsm gene. Finally, a detailed structural comparison shows that the Sm-fold is remarkably well conserved in bacteria, Archaea and Eukarya, and represents a universal and modular building unit for oligomeric RNA binding proteins.     

Target RNA motif and target mRNAs of the Quaking STAR protein

Quaking viable (Qk(v)) mice have developmental defects that result in their characteristic tremor. The quaking (Qk) locus expresses alternatively spliced RNA-binding proteins belonging to the STAR family. To characterize the RNA binding specificity of the QKI proteins, we selected for RNA species that bound QKI from random pools of RNAs and defined the QKI response element (QRE) as a bipartite consensus sequence NACUAAY-N(1-20)-UAAY. A bioinformatic analysis using the QRE identified the three known RNA targets of QKI and 1,430 new putative mRNA targets, of which 23 were validated in vivo. A large proportion of the mRNAs are implicated in development and cell differentiation, as predicted from the phenotype of the Qk(v) mice. In addition, 24% are implicated in cell growth and/or maintenance, suggesting a role for QKI in cancer.     

Structures of the pleiotropic translational regulator Hfq and an Hfq-RNA complex: a bacterial Sm-like protein

In prokaryotes, Hfq regulates translation by modulating the structure of numerous RNA molecules by binding preferentially to A/U-rich sequences. To elucidate the mechanisms of target recognition and translation regulation by Hfq, we determined the crystal structures of the Staphylococcus aureus Hfq and an Hfq-RNA complex to 1.55 and 2.71 A resolution, respectively. The structures reveal that Hfq possesses the Sm-fold previously observed only in eukaryotes and archaea. However, unlike these heptameric Sm proteins, Hfq forms a homo-hexameric ring. The Hfq-RNA structure reveals that the single-stranded hepta-oligoribonucleotide binds in a circular conformation around a central basic cleft, whereby Tyr42 residues from adjacent subunits stack with six of the bases, and Gln8, outside the Sm motif, provides key protein-base contacts. Such binding suggests a mechanism for Hfq function.     

Small RNAs and Hfq capture unfolded RNA target sites during transcription

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Hfq variant with altered RNA binding functions

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS–lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq–RNA interaction but has probably no effect on the folding and the oligomerization of the protein.