Identification of bacterial sRNA regulatory targets using ribosome profiling

Wang, Jing; Rennie, William; Liu, Chaochun; Carmack, C. Steven; Prévost, Karine; Caron, Marie-Pier; Massé, Éric; Ding, Ye; Wade, Joseph T.

doi:10.1093/nar/gkv1158

Cited by 57 publications

(55 citation statements)

References 73 publications

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“…Indeed, for six of the seven sRNAs, the overall change in expression of the set of their bound partners was larger compared to the rest of the genes (p ≤ 0.002 by Kolmogorov-Smirnov test; Figure S4A, right panel for each sRNA). Next, we conducted a similar analysis to test the sRNA effect on the translation of RIL-seq targets, using ribosome profiling data upon change in sRNA expression, available for RyhB (Wang et al., 2015). Indeed, there was a greater change in the ribosome occupancy of RyhB bound partners identified by RIL-seq compared to background genes (p ≤ 1.16 × 10 −8 by Kolmogorov-Smirnov test; Figure S4B, right panel).…”

Section: Resultsmentioning

confidence: 99%

Global Mapping of Small RNA-Target Interactions in Bacteria

et al. 2016

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SummarySmall RNAs (sRNAs) associated with the RNA chaperon protein Hfq are key posttranscriptional regulators of gene expression in bacteria. Deciphering the sRNA-target interactome is an essential step toward understanding the roles of sRNAs in the cellular networks. We developed a broadly applicable methodology termed RIL-seq (RNA interaction by ligation and sequencing), which integrates experimental and computational tools for in vivo transcriptome-wide identification of interactions involving Hfq-associated sRNAs. By applying this methodology to Escherichia coli we discovered an extensive network of interactions involving RNA pairs showing sequence complementarity. We expand the ensemble of targets for known sRNAs, uncover additional Hfq-bound sRNAs encoded in various genomic regions along with their trans encoded targets, and provide insights into binding and possible cycling of RNAs on Hfq. Comparison of the sRNA interactome under various conditions has revealed changes in the sRNA repertoire as well as substantial re-wiring of the network between conditions.

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Section: Resultsmentioning

confidence: 99%

Global Mapping of Small RNA-Target Interactions in Bacteria

et al. 2016

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“…Plasmid variant pKF226-1 ( ydgT-M1::gfp ) was constructed by PCR amplification of pKF127-1 (primer pair JVO-14343/JVO-14344), followed by direct transformation of the linear PCR product into competent E. coli . For plasmids expressing different SdsR mutant variants from the P L promoter, pKF68-3 served as a template for PCR amplification with primer pairs JVO-7161/JVO-7162 (pP L -SdsR C15G; pKF100-1), or JVO-9033/JVO-9034 (pP L -SdsR C38G; pKH6), and self-ligation was carried out as in (22,23). Competent E. coli TOP10 were used for all cloning purposes.…”

Section: Methodsmentioning

confidence: 99%

“…Since Hfq-dependent sRNAs typically operate by short, imperfect ‘seed-pairing’ interactions to regulate multiple targets, the sum of their post-transcriptional activities is expected to affect a large proportion of the mRNAs of these bacteria (20,21). Indeed, some sRNAs such as GcvB and RyhB alone may each regulate ∼1% of all mRNAs expressed in Salmonella or E. coli , respectively (22,23). Nevertheless, the target suites of most sRNAs have not been determined, and it is unclear to which extent these regulators contribute to gene control and microbial physiology.…”

Section: Introductionmentioning

confidence: 99%

The target spectrum of SdsR small RNA inSalmonella

et al. 2016

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Model enteric bacteria such as Escherichia coli and Salmonella enterica express hundreds of small non-coding RNAs (sRNAs), targets for most of which are yet unknown. Some sRNAs are remarkably well conserved, indicating that they serve cellular functions that go beyond the necessities of a single species. One of these ‘core sRNAs’ of largely unknown function is the abundant ∼100-nucleotide SdsR sRNA which is transcribed by the general stress σ-factor, σS and accumulates in stationary phase. In Salmonella, SdsR was known to inhibit the synthesis of the species-specific porin, OmpD. However, sdsR genes are present in almost all enterobacterial genomes, suggesting that additional, conserved targets of this sRNA must exist. Here, we have combined SdsR pulse-expression with whole genome transcriptomics to discover 20 previously unknown candidate targets of SdsR which include mRNAs coding for physiologically important regulators such as the carbon utilization regulator, CRP, the nucleoid-associated chaperone, StpA and the antibiotic resistance transporter, TolC. Processing of SdsR by RNase E results in two cellular SdsR variants with distinct target spectra. While the overall physiological role of this orphan core sRNA remains to be fully understood, the new SdsR targets present valuable leads to determine sRNA functions in resting bacteria.

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“…Through Hfq, sRNAs are capable of pairing with multiple mRNA target sequences, and as a consequence, expression of a single sRNA can exert coordinated regulatory control over an entire set of related genes. For example, in response to iron starvation, expression of the sRNA RyhB downregulates the synthesis of more than 50 proteins involved in iron metabolism and storage (11,12).…”

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confidence: 99%

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

Cameron¹,

Lay

2016

J Bacteriol

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Gene regulation by base pairing between small noncoding RNAs (sRNAs) and their mRNA targets is an important mechanism that allows bacteria to maintain homeostasis and respond to dynamic environments. In Gram-negative bacteria, sRNA pairing and regulation are mediated by several RNA-binding proteins, including the sRNA chaperone Hfq and polynucleotide phosphorylase (PNPase). PNPase and its homolog RNase PH together represent the two 3= to 5= phosphorolytic exoribonucleases found in Escherichia coli; however, the role of RNase PH in sRNA regulation has not yet been explored and reported. Here, we have examined in detail how PNPase and RNase PH interact to support sRNA stability, activity, and base pairing in exponential and stationary growth conditions. Our results indicate that these proteins facilitate the stability and regulatory function of the sRNAs RyhB, CyaR, and MicA during exponential growth. PNPase further appears to contribute to pairing between RyhB and its mRNA targets. During stationary growth, each sRNA responded differently to the absence or presence of PNPase and RNase PH. Finally, our results suggest that PNPase and RNase PH stabilize only Hfq-bound sRNAs. Taken together, these results confirm and extend previous findings that PNPase participates in sRNA regulation and reveal that RNase PH serves a similar, albeit more limited, role as well. These proteins may, therefore, act to protect sRNAs from spurious degradation while also facilitating regulatory pairing with their targets. IMPORTANCEIn many bacteria, Hfq-dependent base-pairing sRNAs facilitate rapid changes in gene expression that are critical for maintaining homeostasis and responding to stress and environmental changes. While a role for Hfq in this process was identified more than 2 decades ago, the identity and function of the other proteins required for Hfq-dependent regulation by sRNAs have not been resolved. Here, we demonstrate that PNPase and RNase PH, the two phosphorolytic RNases in E. coli, stabilize sRNAs against premature degradation and, in the case of PNPase, also accelerate regulation by sRNA-mRNA pairings for certain sRNAs. These findings are the first to demonstrate that RNase PH influences and supports sRNA regulation and suggest shared and distinct roles for these phosphorolytic RNases in this process. Bacteria rapidly alter gene expression through small noncoding RNAs (sRNAs) that act by base pairing with target mRNAs. Although classically identified as important players in bacterial stress response, sRNAs also play general roles regulating the genes involved in cell growth, nutrient acquisition, social behavior, and virulence (1, 2). sRNAs often act through translational repression, in which a given sRNA base pairs with the leader sequence of a target mRNA, obscuring the site that would otherwise form contacts with the ribosomal 30S subunit (3). This pairing can also lead to subsequent RNase E-mediated decay of the mRNA (4). In other cases, pairing with an mRNA leader sequence promotes gene expression by revea...

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Identification of bacterial sRNA regulatory targets using ribosome profiling

Cited by 57 publications

References 73 publications

Global Mapping of Small RNA-Target Interactions in Bacteria

Global Mapping of Small RNA-Target Interactions in Bacteria

The target spectrum of SdsR small RNA inSalmonella

The Phosphorolytic Exoribonucleases Polynucleotide Phosphorylase and RNase PH Stabilize sRNAs and Facilitate Regulation of Their mRNA Targets

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