Coincident Hfq binding and RNase E cleavage sites on mRNA and small regulatory RNAs

Moll, Isabella; Afonyushkin, Taras; Vytvytska, Oresta; Kaberdin, Vladimir R.; Bläsi, Udo

doi:10.1261/rna.5850703

Cited by 242 publications

(249 citation statements)

References 35 publications

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“…Moreover, oligoadenylated 3′ ends are expected to interact with the distal face of Hfq, where the 3′ hydroxyl group is unprotected. Finally, our data indicate that there must yet be additional RNA-binding sites on Hfq that help to bind the sRNA body and that we presume to be responsible for the protection of the body from endonucleolytic cleavage by RNAse E (30,31). It shall be interesting to learn where these additional sites are located and which conformational arrangements within the Hfq/sRNA complex accompany target mRNA binding.…”

Section: The Free 3′-terminal Hydroxyl Group Is Crucial For the High-mentioning

confidence: 83%

Structural basis for RNA 3′-end recognition by Hfq

Sauer

Weichenrieder

2011

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

192

251

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The homohexameric (L)Sm protein Hfq is a central mediator of small RNA-based gene regulation in bacteria. Hfq recognizes small regulatory RNAs (sRNAs) specifically, despite their structural diversity. This specificity could not be explained by previously described RNA-binding modes of Hfq. Here we present a distinct and preferred mode of Hfq-RNA interaction that involves the direct recognition of a uridine-rich RNA 3′ end. This feature is common in bacterial RNA transcripts as a consequence of Rho-independent transcription termination and hence likely contributes significantly to the general recognition of sRNAs by Hfq. Isothermal titration calorimetry shows nanomolar affinity between Salmonella typhimurium Hfq and a hexauridine substrate. We determined a crystal structure of the complex that reveals a constricted RNA backbone conformation in the proximal RNA-binding site of Hfq, allowing for a direct protein contact of the 3′ hydroxyl group. A free 3′ hydroxyl group is crucial for the high-affinity interaction with Hfq also in the context of a full-length sRNA substrate, RybB. The capacity of Hfq to occupy and sequester the RNA 3′ end has important implications for the mechanisms by which Hfq is thought to affect sRNA stability, turnover, and regulation.RNA chaperone | regulation of translation | RNA degradation | prokaryotes H fq is an abundant and widely conserved RNA-binding protein in bacteria and a major player in the RNA-based regulation of gene expression, such as in the adaptive response to cell stress or in the induction of virulence (1, 2).Hfq was originally identified as a host factor in Escherichia coli for the replication of the Qβ phage (3), where it binds to the C-rich 3′ end of plus-strand viral RNA (4). Subsequently, physiological roles of Hfq were described in the regulation of mRNA translation and in mRNA degradation, where it modulates the processing of RNA 3′ ends (1,(5)(6)(7)(8). The most prominent function of Hfq, however, is its interaction with small regulatory RNAs (sRNAs) that act in trans and that are differentially expressed under various metabolic and environmental conditions (9, 10). They globally regulate gene expression via base-pairing to frequently entire sets of partially complementary mRNAs (11,12). Hfq stabilizes sRNAs in the absence of their targets (13). Hfq was also found to facilitate base-pairing to the mRNAs and help trigger subsequent steps, such as the repression of translation and/or the acceleration of decay, but also mRNA activation (11,14). Despite their structural diversity, the recognition of many sRNAs by Hfq is highly specific and even works across species barriers (15). It is an intriguing question how this selectivity is achieved.Crystal structures reveal that bacterial Hfq adopts an (L)Sm fold and forms homohexameric rings, whereas related (L)Sm proteins [Sm proteins and Sm-like (LSm) proteins] in archaea and eukaryotes are found to form homomeric or heteromeric heptamers, respectively (16). Two distinct RNA-binding sites have been described on opposite f...

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Section: The Free 3′-terminal Hydroxyl Group Is Crucial For the High-mentioning

confidence: 83%

Structural basis for RNA 3′-end recognition by Hfq

Sauer

Weichenrieder

2011

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

192

251

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“…One effect of Hfq in vivo is to stabilize sRNAs (25). Thus, possibly DsrA accumulates to higher levels than the other two sRNAs in an hfq mutant.…”

Section: Resultsmentioning

confidence: 99%

Positive regulation by small RNAs and the role of Hfq

Soper

Mandin

Majdalani

et al. 2010

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

258

305

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Bacterial small noncoding RNAs carry out both positive and negative regulation of gene expression by pairing with mRNAs; in Escherichia coli, this regulation often requires the RNA chaperone Hfq. Three small regulatory RNAs (sRNAs), DsrA, RprA, and ArcZ, positively regulate translation of the sigma factor RpoS, each pairing with the 5′ leader to open up an inhibitory hairpin. In vitro, rpoS interaction with sRNAs depends upon an ðAANÞ 4 Hfq-binding site upstream of the pairing region. Here we show that both Hfq and this Hfq binding site are required for RprA or ArcZ to act in vivo and to form a stable complex with rpoS mRNA in vitro; both were partially dispensable for DsrA at 37°C. ArcZ sRNA is processed from 121 nt to a stable 56 nt species that contains the pairing region; only the 56 nt ArcZ makes a strong Hfq-dependent complex with rpoS. For each of these sRNAs, the stability of the sRNA • mRNA complexes, rather than their rate of formation, best predicted in vivo activity. These studies demonstrate that binding of Hfq to the rpoS mRNA is critical for sRNA regulation under normal conditions, but if the stability of the sRNA • mRNA complex is sufficiently high, the requirement for Hfq can be bypassed.Sigma 38 | translational control | Sm-like protein | RNA-protein interactions

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“…The half-life of OxyS in E. coli K12 wild type was reported to be 12-15 min, 14 the half-life in strain MC4100 can create new RNase cleavage sites 18,26 or protect from cleavage. 27,28 While RNase III preferentially cleaves double stranded RNA regions, RNase E recognizes single stranded AU rich segments. 29 To address the role of these RNases in OxyS decay we analyzed RNase deficient strains and compared OxyS stability in the isogenic wild type backgrounds.…”

Section: Resultsmentioning

confidence: 99%