Unknown,Transcriptomics,Genomics,Proteomics

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A small RNA activates CFA synthase by a reprogrammable mechanism of mRNA stabilization

ABSTRACT: Small RNAs use a diversity of well-characterized mechanisms to repress mRNAs, but how they activate gene expression at the mRNA level remains not well understood. The predominant activation mechanism of Hfq-associated small RNAs has been translational control whereby base-pairing with the target prevents the formation of an intrinsic inhibitory structure in the mRNA and promotes translation initiation. Here, we report a translation-independent mechanism whereby the small RNA RydC selectively activates the longer of two isoforms of cfa mRNA (encoding cyclopropane fatty acid synthase) in Salmonella enterica. Target activation is achieved through seed pairing of the pseudoknot-exposed, conserved 5' end of RydC to an upstream region of the cfa mRNA. The seed pairing stabilizes the messenger, likely by interfering directly with RNase E-mediated decay in the 5' untranslated region. Intriguingly, this activation mechanism is fully generic in that it can be reprogrammed to other seed pairing small RNAs, suggesting this mechanism may be utilised in the design of RNA-controlled synthetic circuits. Physiologically, RydC is the first small RNA known to regulate membrane stability. To determine the targets of the small regulatory RNA RydC in S. Typhimurium, we looked at the effect of a short pulse of RydC over-expression on the Salmonella transcriptome. To achieve over-expression, the rydC gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control (also induced by L-arabinose). 2 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674M-bM-^@M-^S1688.

ORGANISM(S): Salmonella enterica subsp. enterica serovar Typhimurium str. SL1344

SUBMITTER: Kai Papenfort

PROVIDER: E-GEOD-48221 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:SgrS RNA is a model for the large class of Hfq-associated small RNAs that act to post-transcriptionally regulate bacterial mRNAs. The function of SgrS is well-characterized in non-pathogenic Escherichia coli where it was originally shown to counteract glucose-phosphate stress by acting as a repressor of ptsG mRNA which encodes the major glucose transporter. We have discovered new SgrS targets in Salmonella Typhimurium, a pathogen related to E. coli, which recently acquired a quarter of all genes by horizontal gene transfer. We demonstrate that the conserved short seed region of SgrS that recognizes ptsG was recruited to target the Salmonella-specific sopD mRNA of a secreted virulence protein. The SgrS-sopD interaction is exceptionally selective; we find that sopD2 mRNA whose gene arose from sopD duplication during Salmonella evolution, is deaf to SgrS owing to a non-productive G:U pair in the potential SgrS-sopD2 RNA duplex versus G:C in SgrS-sopD. In other words, SgrS discriminates the two virulence factor mRNAs at the level of a single hydrogen bond. Our study suggests that bacterial pathogens employ their large suites of conserved Hfq-associated regulators to integrate horizontally acquired genes into existing post-transcriptional networks, just as conserved transcription factors are recruited to tame foreign genes at the DNA level. The results graphically illustrate the importance of the seed regions of bacterial small RNAs to select new targets with high fidelity, and argue that target predictions must consider all-or-none decisions by individual seed nucleotides. To determine the targets of the small regulatory RNA SgrS in S. Typhimurium, we looked at the effect of a short pulse of SgrS over-expression on the Salmonella transcriptome. To achieve over-expression, the sgrS gene was cloned in the pBAD plasmid and induced with 0.2% L-arabinose for 10 min. We then extracted the total RNA for transcriptional profiling. A strain carrying the pBAD plasmid w/o insert was used as negative control. 3 biological replicates were performed. This sRNA target identification strategy has been described in Papenfort et al; Molecular Microbiology (2006) 62(6), 1674–1688.

Project description:The small RNA, ArcZ (previously RyhA/SraH), was discovered in several genome-wide screens in Escherichia coli and Salmonella. Its high degree of genomic conservation, its frequent recovery by shotgun sequencing, and its association with the RNA chaperone, Hfq, identified ArcZ as an abundant enterobacterial âcoreâ small RNA of unknown function. Here, we report that ArcZ acts as a post-transcriptional regulator in Salmonella, repressing the mRNAs of the widely distributed sdaCB (serine uptake) and tpx (oxidative stress) genes, and of STM3216, a horizontally acquired methyl-accepting chemotaxis protein (MCP). Both sdaCB and STM3216 are regulated by sequestration of the ribosome binding site. In contrast, the tpx mRNA is targeted in the coding sequence (CDS), arguing that CDS targeting is more common than appreciated. Transcriptomic analysis of an arcZ deletion strain further argued for the existence of a distinct set of Salmonella loci specifically regulated by ArcZ. In contrast, increased expression of the sRNA altered the steady-state levels of >16% (â¥750) of all Salmonella mRNAs, and rendered the bacteria non-motile. Deep sequencing detected a dramatically changed profile of Hfq-bound sRNAs and mRNAs suggesting that the unprecedented degree of pleiotropic regulation might in part be caused by titration of Hfq binding by ArcZ. This study used three different approaches to identify target genes and biological role of the small RNA ArcZ in Salmonella Typhimurium. Transcriptomic analysis of ArcZ overexpression: Strain JVS-0082 (ÎarcZ) was transformed with plasmids pJV300 (control) and pKP48-1 (parcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptomic analysis of arcZ mutant strain: Strains JVS-007 (WT) and JVS-0082 (ÎarcZ) were transformed with plasmid pJV300 (control) and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture for 6h after cells had reached an OD600=2.0 to attain late stationary phase. Transcriptional effects of ArcZ pulse expression: Strain SL1344 was transformed with plasmids pKP8-35 (pBAD-control) and pKP4-13 (pBAD-ArcZ), and grown in liquid culture (LB broth) inoculated 1:100 from an overnight culture to an OD600 of 1.5. Expression of the insert was induced with L-arabinose (0.2% final concentrations) for 10 min. Three biological replicates were performed for each strain/condition.

Project description:RyhB is a non-coding RNA regulated by the Fur repressor. It has previously been shown to cause the rapid degradation of a number of mRNAs that encode proteins that utilize iron. Here we examine the effect of ectopic RyhB production on global gene expression by microarray analysis. Many of the previously identified targets were found, as well as other mRNAs encoding iron-binding proteins, bringing the total number of regulated operons to at least 18, encoding 56 genes. The two major operons involved in Fe-S cluster assembly showed different behavior; the isc operon appears to be a direct target of RyhB action, while the suf operon does not. This is consistent with previous findings suggesting that the suf genes but not the isc genes are important for Fe-S cluster synthesis under iron-limiting conditions, presumably for essential iron-binding proteins. In addition, we observed repression of Fur-regulated genes upon RyhB expression, interpreted as due to intracellular iron-sparing resulting from reduced synthesis of iron-binding proteins. Our results demonstrate the broad effects of a single non-coding RNA on iron homeostasis. Experiment Overall Design: Derivatives of MG1655 carrying either pBAD-ryhB or the control vector pNM12 were used in all experiments. Experiment Overall Design: The strains used contain the (delta)ara714 allele to prevent catabolism of arabinose and the (delta)ryhB::cat allele to restrict RyhB expression to the inducible pBAD-ryhB vector. Experiment Overall Design: Overnight bacterial cultures were incubated in LB media with ampicillin at a final concentration of 50 ug/mL at 37oC and diluted 1000-fold into 50 mL of fresh LB-ampicillin media at 37oC with agitation. To induce RyhB expression, cultures carrying the pBAD-ryhB construct were grown to an O.D.600 of 0.5 and arabinose was added to the culture at a final concentration of 0.1%. In some experiments, 50 uM FeSO4 was added to the new culture after dilution from overnight culture. Total RNA was extracted from cells at the indicated time using the hot phenol procedure.

Project description:The bacterial heat-shock response is regulated by the alternative sigma factor sigma 32 (RpoH), which responds to misfolded protein stress and directs the RNA polymerase to the promoterss for genes required for protein refolding or degradation. In P. aeruginosa, RpoH is essential for viability under laboratory growth conditions. Here, we used a transcriptomics approach to identify the genes of the RpoH regulon, including RpoH-regulated genes that are essential for P. aeruginosa. We placed the rpoH gene under control of the arabinose inducible PBAD promoter, then deleted the chromosomal rpoH allele. This allowed transcriptomic analysis of the RpoH regulon following a short up-shift in the cellular concentration of RpoH by arabinose addition, in the absence of a sudden increase in temperature. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain grew in the absence of arabinose, indicating that some rpoH expression occurs without arabinose-induction. When arabinose was added, the rpoH mRNA abundance of P. aeruginosa ∆rpoH (PBAD-rpoH) measured by RT-qPCR increased fivefold within 15 min of arabinose addition. Whole genome transcriptome results showed that P. aeruginosa genes required for protein repair or degradation are induced by increased RpoH levels, and that many of the genes induced by RpoH are essential for P. aeruginosa growth. Other stress response genes induced by RpoH are involved in nucleic acid damage and repair and in amino acid metabolism. Annotation of the hypothetical proteins under RpoH control included proteins that may play a role in antibiotic resistances and in non-ribosomal peptide synthesis. The P. aeruginosa ∆rpoH (PBAD-rpoH) strain is impaired in its ability to survive during starvation compared to the wild-type strain. P. aeruginosa ∆rpoH (PBAD-rpoH) also has increased sensitivity to aminoglycoside antibiotics, but not to other classes of antibiotics, whether cultured planktonically or in biofilms. The enhanced aminoglycoside resistance of the mutant strain may be due to indirect effects, such as the build-up of toxic misfolded proteins, or to the direct effect of genes such as aminoglycoside acetyl transferases that are regulated by RpoH. Overall, the results demonstrate that RpoH regulates genes that are essential for viability of P. aeruginosa, that it protects P. aeruginosa from damage from aminoglycoside antibiotics, and that it is required for survival during nutrient limiting conditions. We used Affymetrix microarrays to characterize the RpoH regulon in P. aeruginosa. Using the P. aeruginosa ∆rpoH strain with rpoH under control of the PBAD promoter, we were able to perform transcriptomic analysis of genes induced by a sudden increase (15 min) in the cellular concentration of RpoH, independent from a sudden increase in temperature.

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A small RNA activates CFA synthase by a reprogrammable mechanism of mRNA stabilization

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