Unknown,Transcriptomics,Genomics,Proteomics

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RNA-Seq samples of E. coli K12 MG1655, deletion of ryhB sRNA gene, 2,2’-Dipyridy

ABSTRACT: Bacteria adapt to environmental changes by remodeling their gene expression programs. The change in expression of specific genes is due to the combined effects of various regulators, among them small RNAs (sRNAs). Here we present a straightforward RNA-seq-based approach to infer the contribution of a sRNA to the total change in gene expression levels of specific genes in response to shifts in environmental conditions, demonstrated here for the sRNA RyhB in cells growing under iron limitation stress. We show that the contribution of RyhB to changes in expression levels of specific genes upon iron limitation varies between the genes, from relatively mild contribution to the expression change of some genes to ~100% effect on the expression change of other genes. Interestingly, the contribution of RyhB is high for genes undergoing in total mild expression changes and relatively low for genes undergoing in total high expression changes, implying that for almost all affected genes RyhB renders small changes in expression upon iron limitation. Determining the contribution of a sRNA to the total changes in expression levels of genes in response to an environmental change is important for understanding the integration of regulation by sRNAs in the cellular networks regulating cellular physiology.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Escherichia coli str. K-12 substr. MG1655

SUBMITTER: Meshi Barsheshet

PROVIDER: E-MTAB-10502 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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:Small RNAs (sRNAs) are hypothesized to contribute to hybrid vigor because they maintain genome integrity, contribute to genetic diversity, and control gene expression. We used Illumina sequencing to assess how sRNA populations vary between two maize inbred lines (B73, Mo17) and their hybrid. We sampled sRNAs from the seedling shoot apex and the developing ear, two rapidly growing tissues that program the greater growth of maize hybrids. We found that parental differences in siRNAs primarily originate from repeat regions. Although the maize genome contains greater number and complexity of repeats compared to Arabidopsis or rice, we confirmed that like these simpler plant genomes, 24-nt siRNAs whose abundance differs between maize parents also show a trend of downregulation following hybridization. Surprisingly, hybrid vigor is fully maintained when 24-nt siRNAs are globally reduced by mutation of the RNA-dependent RNA polymerase2 (RDR2) encoded by modifier of paramutation1 (mop1). We also discovered that 21-22nt siRNAs derived from a number of distinct retrotransposon families differentially accumulate between B73 and Mo17 as well as their hybrid. Thus, maize possesses a novel source of genetic variation for regulating both transposons and genes at a genomic scale, which may contribute to its high degree of observed heterosis. sRNA libraries were derived from RNA isolated from the seedling shoot apex and developing ear tissues from B73, Mo17, B73xMo17 and Mo17xB73. The shoot apex was chosen because it is enriched for meristematic tissue where cell proliferation occurs, rates of organ initiation are determined, and organ size is specified. The developing ear was examined because it is enriched in meristematic tissue and is undergoing rapid growth, and also because the mature ear shows the highest degree of heterosis. Total RNA was isolated and separated on a 15% TBE-Urea polyacrylamide gel. Using a 10-bp ladder, the sRNA fraction representing 10-40-bp was excised. sRNA libraries were prepared according to Lu et al. (2007) or manufacturer's instructitions (Illumina). A combination of Perl scripts and FASTX toolkit scripts were used to remove adapters, collapse identical sequences and count reads per sequence. Supplementary processed data text files contain the distinct sRNA sequences for all of the genotypes analyzed in that experiment. Abundance (reads per million) was calculated for each distinct sequence by dividing the number of reads of distinct sRNA in a library by the total number of sRNA reads for that library and multiplying this by 1 million. Genome builds: B73 genome, maizesequence.org release 4a.53 (October, 2009); Mo17 whole genome shotgun clones.

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RNA-Seq samples of E. coli K12 MG1655, deletion of ryhB sRNA gene, 2,2’-Dipyridy

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