Global
            <scp>RNA</scp>
            recognition patterns of post‐transcriptional regulators Hfq and CsrA revealed by
            <scp>UV</scp>
            crosslinking
            <i>in vivo</i>

Holmqvist, Erik; Wright, Patrick R.; Li, Lei; Bischler, Thorsten; Barquist, Lars; Reinhardt, Richard; Backofen, Rolf; Vogel, Jörg

doi:10.15252/embj.201593360

Cited by 285 publications

(377 citation statements)

References 135 publications

(200 reference statements)

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“…Salmonella enterica was reported to somehow activate expression of the uvrY ortholog, sirA, via cAMP-CRP, with positive downstream effects on csrB-lacZ and csrC-lacZ gene fusions (79). In addition, CLIP-seq studies in Salmonella revealed binding of CsrA to crp (cap) leader mRNA at two locations (80), one of which is related in sequence to E. coli BS2 (Fig. 8A).…”

Section: Discussionmentioning

confidence: 99%

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

et al. 2016

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Cyclic AMP (cAMP) and the cAMP receptor protein (cAMP-CRP) and CsrA are the principal regulators of the catabolite repression and carbon storage global regulatory systems, respectively. cAMP-CRP controls the transcription of genes for carbohydrate metabolism and other processes in response to carbon nutritional status, while CsrA binds to diverse mRNAs and regulates translation, RNA stability, and/or transcription elongation. CsrA also binds to the regulatory small RNAs (sRNAs) CsrB and CsrC, which antagonize its activity. The BarA-UvrY two-component signal transduction system (TCS) directly activates csrB and csrC (csrB/C) transcription, while CsrA does so indirectly. We show that cAMP-CRP inhibits csrB/C transcription without negatively regulating phosphorylated UvrY (P-UvrY) or CsrA levels. A crp deletion caused an elevation in CsrB/C levels in the stationary phase of growth and increased the expression of csrB-lacZ and csrClacZ transcriptional fusions, although modest stimulation of CsrB/C turnover by the crp deletion partially masked the former effects. DNase I footprinting and other studies demonstrated that cAMP-CRP bound specifically to three sites located upstream from the csrC promoter, two of which overlapped the P-UvrY binding site. These two proteins competed for binding at the overlapping sites. In vitro transcription-translation experiments confirmed direct repression of csrC-lacZ expression by cAMP-CRP. In contrast, cAMP-CRP effects on csrB transcription may be mediated indirectly, as it bound nonspecifically to csrB DNA. In the reciprocal direction, CsrA bound to crp mRNA with high affinity and specificity and yet exhibited only modest, conditional effects on expression. Our findings are incorporated into an emerging model for the response of Csr circuitry to carbon nutritional status. IMPORTANCECsr (Rsm) noncoding small RNAs (sRNAs) CsrB and CsrC of Escherichia coli use molecular mimicry to sequester the RNA binding protein CsrA (RsmA) away from lower-affinity mRNA targets, thus eliciting major shifts in the bacterial lifestyle. CsrB/C transcription and turnover are activated by carbon metabolism products (e.g., formate and acetate) and by a preferred carbon source (glucose), respectively. We show that cAMP-CRP, a mediator of classical catabolite repression, inhibits csrC transcription by binding to the upstream region of this gene and also inhibits csrB transcription, apparently indirectly. We propose that glucose availability activates pathways for both synthesis and turnover of CsrB/C, thus shaping the dynamics of global signaling in response to the nutritional environment by poising CsrB/C sRNA levels for rapid response.T he Csr (carbon storage regulator) or Rsm (repressor of stationary-phase metabolites) system is a widely conserved bacterial posttranscriptional regulatory system (1-3). Its components, their functions, and the mechanisms by which the central regulator of this system, CsrA or RsmA, affects gene expression have been studied primarily in Gammaproteobacteria (4-6). The...

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Section: Discussionmentioning

confidence: 99%

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

et al. 2016

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“…Peaks were called using JAMM (joint analysis of next-generation sequencing replicates via mixture model clustering) (Ibrahim et al 2015) and PEAKachu (Holmqvist et al 2016). JAMM was designed to report a large number of peaks and relies on external post-processing for peak filtering.…”

Section: Methodsmentioning

confidence: 99%

A mutually exclusive stem–loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila

et al. 2017

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The X chromosome provides an ideal model system to study the contribution of RNA-protein interactions in epigenetic regulation. In male flies, roX long noncoding RNAs (lncRNAs) harbor several redundant domains to interact with the ubiquitin ligase male-specific lethal 2 (MSL2) and the RNA helicase Maleless (MLE) for X-chromosomal regulation. However, how these interactions provide the mechanics of spreading remains unknown. By using the uvCLAP (UV cross-linking and affinity purification) methodology, which provides unprecedented information about RNA secondary structures in vivo, we identified the minimal functional unit of roX2 RNA. By using wild-type and various MLE mutant derivatives, including a catalytically inactive MLE derivative, MLE GET , we show that the minimal roX RNA contains two mutually exclusive stem-loops that exist in a peculiar structural arrangement: When one stem-loop is unwound by MLE, an alternate structure can form, likely trapping MLE in this perpetually structured region. We show that this functional unit is necessary for dosage compensation, as mutations that disrupt this formation lead to male lethality. Thus, we propose that roX2 lncRNA contains an MLE-dependent affinity switch to enable reversible interactions of the MSL complex to allow dosage compensation of the X chromosome.[Keywords: dosage compensation; H4K16ac; MLE; X chromosome; acetylation; roX RNA] Supplemental material is available for this article. RNA helicases are a large family of proteins that have important roles in many aspects of RNA biology. Although the name "helicase" suggests that the main function of these proteins is to destabilize and remove RNA secondary structures, there are several RNA helicases, especially of the DEAD-box subfamily, that use their helicase domains to simply interact with RNA (such as eIF4A3) and not for remodeling. On the other hand, a closely related RNA helicase, eIF4A1, can unwind RNA structures but only when they are relatively weak and short (Rogers et al. 2001). At the other extreme, several helicases are proposed to act as annealers that facilitate the formation of structured RNA rather than the other way around (Jankowsky 2011).Thus, although the helicase family is quite large, specific functions assigned to helicases that involve the actual removal of secondary structures in vivo are rather sparse. Maleless (MLE), known primarily for its role in Drosophila dosage compensation, is an RNA helicase of the DExH family, which is composed of relatively large multidomain helicases that (unlike the DEAD-box family) are thought to work as processive RNA helicases.

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“…One example is the malM 3 ′ UTR, which associates with both Hfq and ProQ via overlapping binding sites (Holmqvist et al 2016;E Holmqvist, in prep.). Given their shared target, the question naturally arises whether ProQ might also interact with Hfq, either through a protein-protein interaction or an RNA-mediated interaction.…”

Section: Proq May Exclude Hfq From Binding To Shared Rna Targetsmentioning

confidence: 99%

Structure of the Escherichia coli ProQ RNA-binding protein

Gonzalez

Hardwick

Maslen

et al. 2017

RNA

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The protein ProQ has recently been identified as a global small noncoding RNA-binding protein in Salmonella, and a similar role is anticipated for its numerous homologs in divergent bacterial species. We report the solution structure of Escherichia coli ProQ, revealing an N-terminal FinO-like domain, a C-terminal domain that unexpectedly has a Tudor domain fold commonly found in eukaryotes, and an elongated bridging intradomain linker that is flexible but nonetheless incompressible. Structure-based sequence analysis suggests that the Tudor domain was acquired through horizontal gene transfer and gene fusion to the ancestral FinO-like domain. Through a combination of biochemical and biophysical approaches, we have mapped putative RNA-binding surfaces on all three domains of ProQ and modeled the protein's conformation in the apo and RNA-bound forms. Taken together, these data suggest how the FinO, Tudor, and linker domains of ProQ cooperate to recognize complex RNA structures and serve to promote RNA-mediated regulation.

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Global RNA recognition patterns of post‐transcriptional regulators Hfq and CsrA revealed by UV crosslinking in vivo

Cited by 285 publications

References 135 publications

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli

A mutually exclusive stem–loop arrangement in roX2 RNA is essential for X-chromosome regulation in Drosophila

Structure of the Escherichia coli ProQ RNA-binding protein

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