Small RNA-Mediated Activation of Sugar Phosphatase mRNA Regulates Glucose Homeostasis

Papenfort, Kai; Sun, Yan; Miyakoshi, Masatoshi; Vanderpool, Carin K.; Vogel, Jörg

doi:10.1016/j.cell.2013.03.003

Cited by 201 publications

(225 citation statements)

References 80 publications

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“…Strains are listed in SI Appendix, Western Blot Analysis. Western blot analyses of GFP and FLAG fusion proteins followed previously published protocols (60). Signals were visualized using an ImageQuant LAS-4000 imager (GE Healthcare) and band intensities were quantified using GelQuant software.…”

Section: Methodsmentioning

confidence: 99%

Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Papenfort¹,

Förstner

Cong³

et al. 2015

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

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Quorum sensing (QS) is a process of cell-to-cell communication that enables bacteria to transition between individual and collective lifestyles. QS controls virulence and biofilm formation in Vibrio cholerae, the causative agent of cholera disease. Differential RNA sequencing (RNA-seq) of wild-type V. cholerae and a locked low-cell-density QS-mutant strain identified 7,240 transcriptional start sites with ∼47% initiated in the antisense direction. A total of 107 of the transcripts do not appear to encode proteins, suggesting they specify regulatory RNAs. We focused on one such transcript that we name VqmR. vqmR is located upstream of the vqmA gene encoding a DNA-binding transcription factor. Mutagenesis and microarray analyses demonstrate that VqmA activates vqmR transcription, that vqmR encodes a regulatory RNA, and VqmR directly controls at least eight mRNA targets including the rtx (repeats in toxin) toxin genes and the vpsT transcriptional regulator of biofilm production. We show that VqmR inhibits biofilm formation through repression of vpsT. Together, these data provide to our knowledege the first global annotation of the transcriptional start sites in V. cholerae and highlight the importance of posttranscriptional regulation for collective behaviors in this human pathogen.Q uorum sensing (QS) is a process of bacterial cell-to-cell communication that relies on the production, release, accumulation, and population-wide detection of extracellular signal molecules called autoinducers. Processes controlled by QS are unproductive when undertaken by an individual bacterium but become effective when performed in unison by the group. In the major human pathogen, Vibrio cholerae, QS orchestrates processes including biofilm formation and virulence factor production (1). In the V. cholerae QS circuit, at low cell density (LCD), LuxO∼P activates transcription of genes encoding four small regulatory RNAs (sRNAs), Qrr1-4, which promote translation of the low cell density master QS regulator, AphA, and repress translation of HapR, the high cell density (HCD) master QS regulator (2, 3). AphA directs the gene expression program for V. cholerae cells to act as individuals. At high cell density, in the presence of autoinducers, LuxO is dephosphorylated and inactivated (4). The Qrr sRNAs are not transcribed, AphA is not produced, and by contrast, the hapR mRNA is translated into HapR protein. HapR controls the gene expression program underpinning collective behaviors (5, 6). Thus, the absence or presence of the Qrr sRNAs directs whether or not V. cholerae engages in QS. The Qrr sRNAs belong to the family of Hfq-binding sRNAs that regulate gene expression through base pairing with target mRNAs (7).An explosion in the discovery of noncoding transcripts, including sRNAs, has occurred due to recently developed transcriptomic technologies such as high-throughput sequencing of cDNAs, e.g., RNA sequencing (RNA-seq). (8). Major advantages of RNA-seq over conventional methods include high sensitivity for low abundance transcripts, ind...

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Section: Methodsmentioning

confidence: 99%

Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Papenfort¹,

Förstner

Cong³

et al. 2015

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

Self Cite

209

265

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“…Following denaturation for 5 min at 95°C, 0.1 OD equivalents of sample were separated on SDS gels. Western blot analyses of σ S , GFP, FLAG fusion proteins, and fluorescence assays followed published protocols (71). Quantitative Western blot data were obtained using a Fuji LAS-3000 imaging system (GE Healthcare), and band intensities were quantified using the AIDA software (Raytest).…”

Section: Methodsmentioning

confidence: 99%

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

Papenfort

Espinosa

Casadesús

et al. 2015

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

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Horizontal gene transfer via plasmid conjugation is a major driving force in microbial evolution but constitutes a complex process that requires synchronization with the physiological state of the host bacteria. Although several host transcription factors are known to regulate plasmid-borne transfer genes, RNA-based regulatory circuits for host-plasmid communication remain unknown. We describe a posttranscriptional mechanism whereby the Hfq-dependent small RNA, RprA, inhibits transfer of pSLT, the virulence plasmid of Salmonella enterica. RprA employs two separate seed-pairing domains to activate the mRNAs of both the sigma-factor σ S and the RicI protein, a previously uncharacterized membrane protein here shown to inhibit conjugation. Transcription of ricI requires σ S and, together, RprA and σ S orchestrate a coherent feedforward loop with AND-gate logic to tightly control the activation of RicI synthesis. RicI interacts with the conjugation apparatus protein TraV and limits plasmid transfer under membrane-damaging conditions. To our knowledge, this study reports the first small RNA-controlled feedforward loop relying on posttranscriptional activation of two independent targets and an unexpected role of the conserved RprA small RNA in controlling extrachromosomal DNA transfer.RprA | sRNA | feedforward control | plasmid conjugation | Hfq

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“…Moreover, in vitro studies with Salmonella and pathogenic E. coli suggest various sRNAs that contribute to pathogenicity. These include the Salmonella pathogenicity island 1 (SPI-1)-associated InvR and DapZ sRNAs, the lipopolysaccharide (LPS) synthesis-controlling MgrR (Moon et al, 2013), or the virulence effector-targeting SgrS sRNA (Papenfort et al, 2013). However, inhibition of many of these well characterized sRNA genes by transposon insertions did not lead to a pronounced growth defect in in vivo models of infection (Barquist et al, 2013;Chaudhuri et al, 2013).…”

Section: Non-coding Rnas Involved In Virulence Infection and Immunitymentioning

confidence: 99%

Dual RNA-seq of pathogen and host

2012

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SummaryThe infection of a eukaryotic host cell by a bacterial pathogen is one of the most intimate examples of cross-kingdom interactions in biology. Infection processes are highly relevant from both a basic research as well as a clinical point of view. Sophisticated mechanisms have evolved in the pathogen to manipulate the host response and vice versa host cells have developed a wide range of anti-microbial defense strategies to combat bacterial invasion and clear infections.However, it is this diversity and complexity that makes infection research so challenging to technically address as common approaches have either been optimized for bacterial or eukaryotic organisms. Instead, methods are required that are able to deal with the often dramatic discrepancy between host and pathogen with respect to various cellular properties and processes. One class of cellular macromolecules that exemplify this host-pathogen heterogeneity is given by their transcriptomes: Bacterial transcripts differ from their eukaryotic counterparts in many aspects that involve both quantitative and qualitative traits. The entity of RNA transcripts present in a cell is of paramount interest as it reflects the cell's physiological state under the given condition. Genome-wide transcriptomic techniques such as RNA-seq have therefore been used for single-organism analyses for several years, but their applicability has been limited for infection studies.The present work describes the establishment of a novel transcriptomic approach for infection biology which we have termed "Dual RNA-seq". Using this technology, it was intended to shed light particularly on the contribution of non-protein-encoding transcripts to virulence, as these classes have mostly evaded previous infection studies due to the lack of suitable methods. The performance of Dual RNA-seq was evaluated in an in vitro infection model based on the important facultative intracellular pathogen Salmonella enterica serovar Typhimurium and different human cell lines. Dual RNA-seq was found to be capable of capturing all major bacterial and human transcript classes and proved reproducible. During the course of these experiments, a previously largely uncharacterized bacterial small non-coding RNA (sRNA), referred to as STnc440, was identified as one of the most strongly induced genes in intracellular Salmonella.Interestingly, while inhibition of STnc440 expression has been previously shown to cause a virulence defect in different animal models of Salmonellosis, the underlying molecular mechanisms have remained obscure. Here, classical genetics, transcriptomics and biochemical assays proposed a complex model of Salmonella gene expression control that is orchestrated by this sRNA. In particular, STnc440 was found to be involved in the regulation of multiple bacterial target mRNAs by direct base pair interaction with consequences for Salmonella virulence and

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Small RNA-Mediated Activation of Sugar Phosphatase mRNA Regulates Glucose Homeostasis

Cited by 201 publications

References 80 publications

Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Differential RNA-seq of Vibrio cholerae identifies the VqmR small RNA as a regulator of biofilm formation

Small RNA-based feedforward loop with AND-gate logic regulates extrachromosomal DNA transfer in Salmonella

Dual RNA-seq of pathogen and host

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