Competition among Hfq-binding small RNAs in Escherichia coli

A major class of small bacterial RNAs (sRNAs) regulate translation and mRNA stability by pairing with target mRNAs, dependent upon the RNA chaperone Hfq. Hfq, related to the Lsm/Sm families of splicing proteins, binds the sRNAs and stabilizes them in vivo and stimulates pairing with mRNAs in vitro. Although Hfq is abundant, the sRNAs, when induced, are similarly abundant. Therefore, Hfq may be limiting for sRNA function. We find that, when overexpressed, a number of sRNAs competed with endogenous sRNAs for binding to Hfq. This correlated with lower accumulation of the sRNAs (presumably a reflection of the loss of Hfq binding), and lower activity of the sRNAs in regulating gene expression. Hfq was limiting for both positive and negative regulation by the sRNAs. In addition, deletion of the gene for an expressed and particularly effective competitor sRNA improved the regulation of genes by other sRNAs, suggesting that Hfq is limiting during normal growth conditions. These results support the existence of a hierarchy of sRNA competition for Hfq, modulating the function of some sRNAs.     

Essential requirements for robust signaling in Hfq dependent small RNA networks

Bacteria possess networks of small RNAs (sRNAs) that are important for modulating gene expression. At the center of many of these sRNA networks is the Hfq protein. Hfq's role is to quickly match cognate sRNAs and target mRNAs from among a large number of possible combinations and anneal them to form duplexes. Here we show using a kinetic model that Hfq can efficiently and robustly achieve this difficult task by minimizing the sequestration of sRNAs and target mRNAs in Hfq complexes. This sequestration can be reduced by two non-mutually exclusive kinetic mechanisms. The first mechanism involves heterotropic cooperativity (where sRNA and target mRNA binding to Hfq is influenced by other RNAs bound to Hfq); this cooperativity can selectively decrease singly-bound Hfq complexes and ternary complexes with non-cognate sRNA-target mRNA pairs while increasing cognate ternary complexes. The second mechanism relies on frequent RNA dissociation enabling the rapid cycling of sRNAs and target mRNAs among different Hfq complexes; this increases the probability the cognate ternary complex forms before the sRNAs and target mRNAs degrade. We further demonstrate that the performance of sRNAs in isolation is not predictive of their performance within a network. These findings highlight the importance of experimentally characterizing duplex formation in physiologically relevant contexts with multiple RNAs competing for Hfq. The model will provide a valuable framework for guiding and interpreting these experiments.     

Paradoxical suppression of small RNA activity at high Hfq concentrations due to random-order binding

Small RNAs (sRNAs) are important regulators of gene expression during bacterial stress and pathogenesis. sRNAs act by forming duplexes with mRNAs to alter their translation and degradation. In some bacteria, duplex formation is mediated by the Hfq protein, which can bind the sRNA and mRNA in each pair in a random order. Here we investigate the consequences of this random-order binding and experimentally demonstrate that it can counterintuitively cause high Hfq concentrations to suppress rather than promote sRNA activity in Escherichia coli. As a result, maximum sRNA activity occurs when the Hfq concentration is neither too low nor too high relative to the sRNA and mRNA concentrations (‘Hfq set-point’). We further show with models and experiments that random-order binding combined with the formation of a dead-end mRNA–Hfq complex causes high concentrations of an mRNA to inhibit its own duplex formation by sequestering Hfq. In such cases, maximum sRNA activity requires an optimal mRNA concentration (‘mRNA set-point’) as well as an optimal Hfq concentration. The Hfq and mRNA set-points generate novel regulatory properties that can be harnessed by native and synthetic gene circuits to provide greater control over sRNA activity, generate non-monotonic responses and enhance the robustness of expression.     

Global analysis of small RNA and mRNA targets of Hfq

Hfq, a bacterial member of the Sm family of RNA-binding proteins, is required for the action of many small regulatory RNAs that act by basepairing with target mRNAs. Hfq binds this family of small RNAs efficiently. We have used co-immunoprecipitation with Hfq and direct detection of the bound RNAs on genomic microarrays to identify members of this small RNA family. This approach was extremely sensitive; even Hfq-binding small RNAs expressed at low levels were readily detected. At least 15 of 46 known small RNAs in E. coli interact with Hfq. In addition, high signals in other intergenic regions suggested up to 20 previously unidentified small RNAs bind Hfq; five were confirmed by Northern analysis. Strong signals within genes and operons also were detected, some of which correspond to known Hfq targets. Within the argX-hisR-leuT-proM operon, Hfq appears to compete with RNase E and modulate RNA processing and degradation. Thus Hfq immunoprecipitation followed by microarray analysis is a highly effective method for detecting a major class of small RNAs as well as identifying new Hfq functions.     

Major role for mRNA binding and restructuring in sRNA recruitment by Hfq

Bacterial small RNAs (sRNAs) modulate gene expression by base-pairing with target mRNAs. Many sRNAs require the Sm-like RNA binding protein Hfq as a cofactor. Well-characterized interactions between DsrA sRNA and the rpoS mRNA leader were used to understand how Hfq stimulates sRNA pairing with target mRNAs. DsrA annealing stimulates expression of rpoS by disrupting a secondary structure in the rpoS leader, which otherwise prevents translation. Both RNAs bind Hfq with similar affinity but interact with opposite faces of the Hfq hexamer. Using mutations that block interactions between two of the three components, we demonstrate that Hfq binding to a functionally critical (AAN)(4) motif in rpoS mRNA rescues DsrA binding to a hyperstable rpoS mutant. We also show that Hfq cannot stably bridge the RNAs. Persistent ternary complexes only form when the two RNAs are complementary. Thus, Hfq mainly acts by binding and restructuring the rpoS mRNA. However, Hfq binding to DsrA is needed for maximum annealing in vitro, indicating that transient interactions with both RNAs contribute to the regulatory mechanism.     

Hfq proximity and orientation controls RNA annealing

Regulation of bacterial gene networks by small non-coding RNAs (sRNAs) requires base pairing with messenger RNA (mRNA) targets, which is facilitated by Hfq protein. Hfq is recruited to sRNAs and mRNAs through U-rich- and A-rich-binding sites, respectively, but their distance from the sRNA–mRNA complementary region varies widely among different genes. To determine whether distance and binding orientation affect Hfq’s chaperone function, we engineered ‘toy’ RNAs containing strong Hfq-binding sites at defined distances from the complementary target site. We show that RNA annealing is fastest when the distal face of Hfq binds an A-rich sequence immediately 3′ of the target. This recruitment advantage is lost when Hfq binds >20 nt away from the target, but is partially restored by secondary structure that shortens this distance. Although recruitment through Hfq’s distal face accelerates RNA annealing, tight binding of six Us to Hfq’s proximal face inhibits annealing. Finally, we show that ectopic A-rich motifs dramatically accelerate base pairing between DsrA sRNA and a minimal rpoS mRNA in the presence of Hfq, demonstrating that proximity and orientation predict the activity of Hfq on long RNAs.