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

Soper, Toby J.; Doxzen, Kevin W.; Woodson, Sarah A.

doi:10.1261/rna.2767211

Cited by 67 publications

(109 citation statements)

References 26 publications

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“…The annealing kinetics between the molecular beacon (50 nM) and target RNA (100 nM) in the presence of 0-250 nM Hfq hexamer in TNK buffer at 30°C was measured by stopped flow fluorescence spectroscopy as described previously (52,66) and in SI Materials and Methods. MgCl 2 was not included in the annealing buffer, because it is not required for annealing or Hfq binding and because it can lead to RNA aggregation (15).…”

Section: Methodsmentioning

confidence: 99%

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

Santiago-Frangos

Kumari

Schu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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149

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The bacterial Sm protein and RNA chaperone Hfq stabilizes small noncoding RNAs (sRNAs) and facilitates their annealing to mRNA targets involved in stress tolerance and virulence. Although an arginine patch on the Sm core is needed for Hfq's RNA chaperone activity, the function of Hfq's intrinsically disordered C-terminal domain (CTD) has remained unclear. Here, we use stopped flow spectroscopy to show that the CTD of Escherichia coli Hfq is not needed to accelerate RNA base pairing but is required for the release of dsRNA. The Hfq CTD also mediates competition between sRNAs, offering a kinetic advantage to sRNAs that contact both the proximal and distal faces of the Hfq hexamer. The change in sRNA hierarchy caused by deletion of the Hfq CTD in E. coli alters the sRNA accumulation and the kinetics of sRNA regulation in vivo. We propose that the Hfq CTD displaces sRNAs and annealed sRNA·mRNA complexes from the Sm core, enabling Hfq to chaperone sRNA-mRNA interactions and rapidly cycle between competing targets in the cell.A member of the Sm protein family, Hfq was first identified as a host factor for phage Q beta. Hfq is found in at least 50% of sequenced bacterial genomes (1) and, in many bacteria, contributes to posttranscriptional regulation by small noncoding RNAs (sRNAs). Deletion of Hfq leads to pleiotropic effects, such as altered cellular morphology, slow growth, maladaptation to stress, and avirulence (2-5).Escherichia coli Hfq comprises an Sm domain (amino acids 7-66) that assembles into a stable hexameric ring and an intrinsically disordered C-terminal domain (CTD) that projects from the rim of the hexamer (6-10). The Sm ring binds to both sRNAs and target mRNAs, stabilizing the sRNAs against turnover (11-13) and facilitating base pairing with complementary sequences in the mRNA (7,14,15). The conserved "proximal" face of the Hfq hexamer interacts with uridines at the sRNA 3′ end (9, 16, 17), whereas the "distal" face of Hfq binds AAN triplet repeats (9, 16, 17) often found in the 5′ UTRs of target mRNAs. These sequence-specific interactions recruit sRNAs and mRNAs to Hfq, allowing arginine-rich patches along the rim of Hfq to catalyze base pairing between complementary strands (18).Although the functional importance of the Sm domain is established, the function of the disordered CTD has been unclear. The Hfq CTD varies greatly in length and sequence composition between bacterial families (1, 19), ranging from 7-residue stubs in Bacillaceae (20) to 100-residue tails in Moraxellaceae (21, 22) (Fig. S1). Previous studies reached conflicting conclusions about the importance of the Hfq CTD for sRNA regulation. In early studies, C-terminal deletions of hfq had no obvious phenotype in E. coli (23, 24) and little effect on sRNA binding (23,25). By contrast, later studies found that the CTD was required for in vitro annealing and proper regulation by sRNAs and normal binding to long RNAs, such as the rpoS mRNA (19,26,27). Moreover, Hfq from Pseudomonas aeruginosa and Clostridium difficile, which have much shor...

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Section: Methodsmentioning

confidence: 99%

C-terminal domain of the RNA chaperone Hfq drives sRNA competition and release of target RNA

Santiago-Frangos

Kumari

Schu

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

149

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“…Although the crystal structures of S. aureus Hfq associated with short RNA sequences were the first to be solved (Brennan and Link, 2007), its function still remains mysterious. The protein adopts an homohexamer structure organized into a doughnut with a proximal and distal faces recognizing unpaired uridine-rich and adenine-rich sequences, respectively (e.g., Brennan and Link, 2007;Horstmann et al, 2012;Link et al, 2009;Mikulecky et al, 2004;Someya et al, 2012;Soper et al, 2011). In enterobacteriaceae, the proximal and distal faces of the Hfq hexamer specifically bind sRNA and mRNA targets.…”

Section: Hfq Protein a Mysterious Protein In S Aureusmentioning

confidence: 99%

The importance of regulatory RNAs in Staphylococcus aureus

Tomasini

François

Howden

et al. 2014

Infection, Genetics and Evolution

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a b s t r a c t RNA molecules with regulatory functions in pathogenic bacteria have benefited from a renewed interest these two last decades. In Staphylococcus aureus, recent genome-wide approaches have led to the discovery that almost 10-20% of genes code for RNAs with critical regulatory roles in adaptive processes. These RNAs include trans-acting RNAs, which mostly act through binding to target mRNAs, and cis-acting RNAs, which include regulatory regions of mRNAs responding to various metabolic signals. Besides recent analysis of S. aureus transcriptome has revealed an unprecedented existence of pervasive transcription generating a high number of weakly expressed antisense RNAs along the genome as well as numerous mRNAs with overlapped regions. Here, we will illustrate the diversity of trans-acting RNAs and illustrate how they are integrated into complex regulatory circuits, which link metabolism, stress response and virulence.

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“…29 In addition, some studies addressing the requirement for sRNAs and/or Hfq were performed at 37 °C, 24 where DsrA is less abundant than at 24 °C 16,17 ( Fig. 1, lanes 2 and 4).…”

Section: Resultsmentioning

confidence: 99%

“…27,28 Moreover, Hfq binding to an (AAN) 4 repeat element in the rpoS leader has been shown to be critical for regulation by sRNAs, 24 and for DsrA·rpoS duplex formation. 29 Binding to these A-rich motifs has been suggested to result in restructuring of the rpoS leader, priming the mRNA for productive interactions with DsrA. 29 Despite intensive research on the DsrA-rpoS model system, the Hfq-dependent steps toward ribosome loading on rpoS mRNA at low temperature are not completely understood.…”

Section: Introductionmentioning

confidence: 99%

“…29 Binding to these A-rich motifs has been suggested to result in restructuring of the rpoS leader, priming the mRNA for productive interactions with DsrA. 29 Despite intensive research on the DsrA-rpoS model system, the Hfq-dependent steps toward ribosome loading on rpoS mRNA at low temperature are not completely understood. Here, we have addressed in vivo whether DsrA·rpoS annealing is sufficient for efficient RpoS synthesis at low temperature.…”

Section: Introductionmentioning

confidence: 99%

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