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C-Terminal Domain of the RNA Chaperone Hfq Drives Srna

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C-Terminal Domain of the RNA Chaperone Hfq Drives Srna C-terminal domain of the RNA chaperone Hfq drives PNAS PLUS sRNA competition and release of target RNA Andrew Santiago-Frangosa, Kumari Kavitab, Daniel J. Schub,1, Susan Gottesmanb,2, and Sarah A. Woodsonc,2 aCell, Molecular, Developmental Biology, and Biophysics Program, Johns Hopkins University, Baltimore, MD 21218; bLaboratory of Molecular Biology, National Cancer Institute, Bethesda, MD 20892; and cThomas C. Jenkins Department of Biophysics, Johns Hopkins University, Baltimore, MD 21218 Contributed by Susan Gottesman, August 9, 2016 (sent for review May 12, 2016; reviewed by Kathleen B. Hall and E. Gerhart H. Wagner) The bacterial Sm protein and RNA chaperone Hfq stabilizes small This variability suggested to us that the CTD either serves an noncoding RNAs (sRNAs) and facilitates their annealing to mRNA autoregulatory function or modulates sRNA regulation in different targets involved in stress tolerance and virulence. Although an argi- bacteria (30). As a result, the apparent importance of the CTD may nine patch on the Sm core is needed for Hfq’s RNA chaperone activ- depend on the sRNA being studied. This possibility became more ity, the function of Hfq’s intrinsically disordered C-terminal domain apparent with the recognition that E. coli sRNAs can be divided (CTD) has remained unclear. Here, we use stopped flow spectros- into two classes based on their interactions with Hfq (31). The copy to show that the CTD of Escherichia coli Hfq is not needed to common Class I sRNAs typically bind the proximal face and rim of accelerate RNA base pairing but is required for the release of dsRNA. Hfq (14, 32–34), whereas a smaller number of Class II sRNAs The Hfq CTD also mediates competition between sRNAs, offering a contact the distal face of Hfq via an AAN motif near the 5′ end of kinetic advantage to sRNAs that contact both the proximal and dis- thesRNAinadditiontotheproximalface(31,35).TheCTDisnot tal faces of the Hfq hexamer. The change in sRNA hierarchy caused essential for sRNA recognition, because truncated Hfq variants by deletion of the Hfq CTD in E. coli alters the sRNA accumulation lacking all or part of the CTD bind sRNAs in vitro and in the cell and the kinetics of sRNA regulation in vivo. We propose that the Hfq (10, 23–26, 28, 36). However, partial ordering of the CTDs in a CTD displaces sRNAs and annealed sRNA·mRNA complexes from the recent structure of Hfq bound to RydC sRNA suggested that the Sm core, enabling Hfq to chaperone sRNA–mRNA interactions and CTDs help recruit sRNAs to Hfq (36). rapidly cycle between competing targets in the cell. To understand how the disordered CTD modulates Hfq’s chaperone function, we compared the RNA binding and anneal- small noncoding RNA | RNA chaperone | intrinsically disordered protein | ing activities of full-length E. coli Hfq (Hfq102) with a truncated posttranscriptional regulation | ChiX protein lacking the entire CTD (Hfq65). Stopped flow spectros- copy and fluorescence anisotropy results show that the CTD is not member of the Sm protein family, Hfq was first identified as required for RNA annealing as previously thought (19, 26) but Aa host factor for phage Q beta. Hfq is found in at least 50% rather, is needed for dissociation of the dsRNA product. We of sequenced bacterial genomes (1) and, in many bacteria, con- further show that the CTD enables kinetic competition between tributes to posttranscriptional regulation by small noncoding RNAs sRNAs, which can actively displace each other from Hfq (37). (sRNAs). Deletion of Hfq leads to pleiotropic effects, such as al- Deleting the hfq CTD in E. coli disrupts the hierarchy of sRNA tered cellular morphology, slow growth, maladaptation to stress, accumulation and alters the kinetics of sRNA–mRNA interac- and avirulence (2–5). tions. We propose that the CTDs modulate Hfq’s chaperone ac- Escherichia coli Hfq comprises an Sm domain (amino acids tivity by stimulating the cycling of sRNAs and sRNA·mRNA 7–66) that assembles into a stable hexameric ring and an intrin- complexes on the Sm ring. sically disordered C-terminal domain (CTD) that projects from the rim of the hexamer (6–10). The Sm ring binds to both sRNAs Significance and target mRNAs, stabilizing the sRNAs against turnover (11–13) and facilitating base pairing with complementary sequences in the The RNA chaperone Hfq binds hundreds of small noncoding mRNA (7, 14, 15). The conserved “proximal” face of the Hfq RNAs (sRNAs) and facilitates their interactions with mRNAs, hexamer interacts with uridines at the sRNA 3′ end (9, 16, 17), regulating bacterial stress responses and virulence. Hfq is limit- whereas the “distal” face of Hfq binds AAN triplet repeats ing in the cell and must release RNAs after they base pair. Most (9, 16, 17) often found in the 5′ UTRs of target mRNAs. These bacterial Hfqs contain an intrinsically disordered C-terminal do- sequence-specific interactions recruit sRNAs and mRNAs to main (CTD), with unknown function. Time-resolved assays now Hfq, allowing arginine-rich patches along the rim of Hfq to catalyze show that CTDs are needed to displace base-paired RNA, recy- cling Hfq. The CTDs also enable kinetic competition between base pairing between complementary strands (18). different sRNAs in Escherichia coli, allowing some sRNAs to Although the functional importance of the Sm domain is estab- bind Hfq and accumulate, whereas other sRNAs are degraded. lished, the function of the disordered CTD has been unclear. The We propose that the CTDs sweep sRNAs from the surface of Hfq CTD varies greatly in length and sequence composition between Hfq. This displacement allows Hfq to search among potential bacterial families (1, 19), ranging from 7-residue stubs in Bacillaceae RNA partners and establishes a hierarchy of sRNA regulation. (20) to 100-residue tails in Moraxellaceae (21, 22) (Fig. S1). Previous studies reached conflicting conclusions about the importance of the Author contributions: A.S.-F., K.K., D.J.S., S.G., and S.A.W. designed research; A.S.-F., K.K., Hfq CTD for sRNA regulation. In early studies, C-terminal dele- and D.J.S. performed research; A.S.-F., K.K., D.J.S., S.G., and S.A.W. analyzed data; and BIOPHYSICS AND tions of hfq had no obvious phenotype in E. coli (23, 24) and little A.S.-F., K.K., S.G., and S.A.W. wrote the paper. COMPUTATIONAL BIOLOGY effect on sRNA binding (23, 25). By contrast, later studies found that Reviewers: K.B.H., Washington University Medical School; and E.G.H.W., Uppsala University. the CTD was required for in vitro annealing and proper regulation The authors declare no conflict of interest. 1 by sRNAs and normal binding to long RNAs, such as the rpoS Present address: Center for Drug Evaluation and Research, Food and Drug Administra- tion, Silver Spring, MD 20993-0002. mRNA (19, 26, 27). Moreover, Hfq from Pseudomonas aeruginosa 2To whom correspondence may be addressed. Email: [email protected] or swoodson@ and Clostridium difficile, which have much shorter CTDs than E. coli jhu.edu. · Hfq, functionally replace E. coli Hfq for certain sRNA mRNA pairs This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. but not others (28, 29). 1073/pnas.1613053113/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1613053113 PNAS | Published online September 28, 2016 | E6089–E6096 Downloaded by guest on September 27, 2021 Table 1. Equilibrium dissociation constants for Hfq By contrast, sRNA stem loops interacted more strongly with the rim of Hfq when the CTD was deleted. We designed a min- RNA (Hfq102)6,nM (Hfq65)6,nM imal rim binding stem loop “minRCRB” containing a 4-nt 5′- DsrA* 42.9 ± 6.0 75.0 ± 8.0 CUUC overhang derived from RydC and a 5-nt loop derived RyhB* 40.3 ± 6.9 93.5 ± 19.5 from RybB. Surprisingly, minRCRB bound Hfq65 twofold more RprA* 42.2 ± 1.3 68.7 ± 3.2 tightly than Hfq102 (Table 1). minRCRB primarily binds the rim ChiX* 36.2 ± 3.1 43.9 ± 10.2 of Hfq as intended, because the Hfq rim mutation R16A raised † A18-FAM 7.4 ± 0.3 8.8 ± 1.1 the K for minRCRB above 170 nM for Hfq65 . This rim mu- † d 6 D16-FAM 15.5 ± 0.9 20.0 ± 1.3 tation also raised the K 10-fold for synthetic molecular beacon † d minRCRB 13.9 ± 1.5 6.46 ± 0.7 † (Fig. 1), which is closed by a 5-bp stem. The molecular beacon Beacon 118 ± 22 14.5 ± 3.2 also exhibited an eightfold preference for Hfq65 compared with Values are the mean ± SD of three independent experiments. full-length Hfq102 (Table 1). These results indicated that the *Kd values for sRNAs were determined by EMSAs at 30 °C in 10 mM Tris-HCl, CTDs inhibit interactions between RNA stem loops and the rim pH 7.5, 50 mM NaCl, 50 mM KCl. of Hfq. † Kd values for oligoribonucleotides were determined by fluorescence anisotropy. Hfq CTD Lowers the RNA Annealing Rate. To delineate how the intrinsically disordered CTDs modulate Hfq’s chaperone func- tion, we compared the RNA annealing activity of full-length Results Hfq102 and truncated Hfq65. We measured base pairing be- Hfq CTD Weakens Binding of RNA Stem Loops to the Rim of Hfq. To tween a fluorescent RNA molecular beacon and a 16-nt Target determine how the CTD affects RNA interactions with different RNA that is complementary to the loop of the molecular beacon surfaces of Hfq, we compared the affinities of four sRNAs and but lacks a specific binding site for Hfq (41).
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