Similar Datasets
Project description:Expression of E. coli RNase toxins
| PRJNA602534 | ENA
Project description:Identification of RNase III cleavage sites in Escherichia coli
| PRJEB26394 | ENA
Project description:In this study, we performed RNA-sequencing on an E. coli model system to confirm known sites, identify novel targets, and determine the impact of RNase III cleavage events on transcript degradation and metabolic phenotypes. To find cleavage sites, we compared the abundance of sequencing reads across the transcriptome of a wild-type E. coli and an rnc- deletion mutant. The RNA-sequencing approach provided wider coverage and unprecedented resolution of mRNA abundance at each position in the transcriptome compared to prior studies that used qPCR, Northern blots, or microarrays. In addition to data collected from exponentially growing cells, we observed the effects of RNase III cleavage on transcript degradation by collecting samples in a time course after stopping nascent transcription with rifampicin. This work was supported by the Department of Energy Grant DE-SC0010329
2017-02-24 | GSE95318 | GEO
Project description:In this study we generated 5'P libraries in wt and RNAse III mutant strains, grown to exponential and stationary phases. Libraries that retain short RNA fragments were also generated in both growth phases. After sequencing by Illumina NextSeq 500 system, reads were mapped to E. coli genome NC_000913.3. By comparing the read start counts per position in the wt and mutant strain libraries we identified the cleavage sites of RNase III.
2018-09-30 | E-MTAB-6704 | biostudies-arrayexpress
Project description:The purpose of this study was to identify the RNA cleavage targets and specificities of 9 endoribonuclease toxins from toxin-antitoxin systems in E. coli MG1655.
2021-07-13 | GSE179607 | GEO
Project description:Small RNAs (sRNAs) are major regulators of gene expression in bacteria. Traditionally, sRNAs were considered mainly as regulators of translation, exerting their regulatory function by base pairing with their target mRNAs, and through this, blocking or exposing the ribosome binding site. However, accumulating evidence suggest that sRNAs not only regulate translation, but also affect the transcript stability by assisting or interfering with endoribonuclesaes. While cooperation of sRNAs with endoribonuclesaes was demonstrated for a few genes, its extent in the bacterial cell was not assessed in large scale. Here, we take advantage of large-scale RNA- seq-based approaches to study the extent of cooperation between sRNAs with the major endoribonuclease in Escherichia coli, RNase E. As a model sRNA, we use the well-known sRNA GcvB, a sRNA that regulates genes involved in amino acid metabolism and transport. To study the cooperation between GcvB and RNase E we apply the mutant cycle approach to four strains of E. coli: wt; GcvB mutant/RNase E wt; GcvB wt/RNase E mutant; GcvB mutant/RNase E mutant. By applying RNA-seq and differential gene expression analysis, we infer different modes of cooperation between RNase E and GcvB, and show that a statistically significant number of GcvB targets are downregulated by cooperation between the two regulators. Furthermore, using the TIER-seq approach to map RNase E cleavage sites transcriptome-wide and analysing them in view of GcvB binding sites, we attempt to infer the mechanisms of cooperation between the two regulators.
2024-05-12 | E-MTAB-12466 | biostudies-arrayexpress
Project description:Abstract: Ribonuclease E (RNase E) has a key role in mRNA degradation and the processing of catalytic and structural RNAs in E. coli. We report the discovery of an evolutionarily conserved 17.4 kDa protein, here named RraA (regulator of ribonuclease activity A) that binds to RNase E and inhibits RNase E endonucleolytic cleavages without altering cleavage site specificity or interacting detectably with substrate RNAs. Overexpression of RraA circumvents the effects of an autoregulatory mechanism that normally maintains the RNase E cellular level within a narrow range, resulting in the genome-wide accumulation of RNase E-targeted transcripts. While not required for RraA action, the C-terminal RNase E region that serves as a scaffold for formation of a multiprotein degradosome complex modulates the inhibition of RNase E catalytic activity by RraA. Our results reveal a possible mechanism for the dynamic regulation of RNA decay and processing by inhibitory RNase binding proteins. This SuperSeries is composed of the SubSeries listed below.
2006-03-08 | GSE4383 | GEO
Project description:Members of the ribonuclease (RNase) III family regulate gene expression by processing double-stranded (ds) RNA. The founding member of the family, Escherichia coli (Ec) RNase III, is the most comprehensively studied and its E38A mutant (EcE38A) is an economical reagent for the preparation of small interfering (si) RNA cocktails. Previously, it was shown that EcRNase III recognizes dsRNA with little specificity and that EcE38A mainly produces 23-nucleotide (nt) siRNAs. To characterize substrate specificity and product size, we performed in vitro cleavage of dsRNAs by bacterial RNase IIIs and delineated the cleavage products by next generation sequencing. Surprisingly, we found that RNase III cleaves dsRNA at preferred sites and most siRNAs produced by EcE38A are 22 nt long. We eliminated the sequence specificity of EcE38A through the introduction of additional mutations, thereby creating a reagent that is ideally suited for producing heterogeneous siRNA cocktails to be used in gene silencing studies.
2019-05-26 | GSE120052 | GEO
Project description:The Escherichia coli endoribonucleases RNase E (Rne) and RNase G (Rng) have sequence similarity and broadly similar sequence specificity. Whereas the absence of Rne normally is lethal, we show here that E. coli bacteria that lack the rne gene can be made viable by overexpression of Rng. Rng-complemented cells accumulated precursors of 5S ribosomal RNA (rRNA) and the RNA component of RNase P (i.e. M1 RNA), indicating that normal processing of these Rne-cleaved RNAs was not restored by RNase G; additionally, neither 5S rRNA nor M1 RNA was generated from precursors by RNase G cleavage in vitro. Using DNA microarrays containing 4405 Escherichia coli open reading frames (ORFs), we identified mRNAs whose steady-state level was affected by Rne, Rng or the N-terminal catalytic domain of RNase E. Most transcript species affected by RNase E deficiency were also elevated in an rne deletion mutant complemented by Rng. However, approximately 100 mRNAs that accumulated in Rne-deficient cells were decreased by rng-complemention, thus identifying targets whose processing or degradation may be the basis for RNase E essentiality. Remarkably prominent in this group were mRNAs implicated in energy-generating pathways or in the synthesis or degradation of macromolecules. Set of arrays that are part of repeated experiments Keywords: Biological Replicate
2005-08-13 | GSE3123 | GEO
Project description:Bacterial toxin-antitoxin systems help bacteria to reduce their metabolism in various stressful conditions and also play a part in generating antibiotic tolerant bacterial subpopulation called persisters. Many toxins of the bacterial toxin-antitoxin systems are ribonucleases. Such toxins have been mostly viewed as degraders of mRNA, however recently it was demonstrated that they are also capable of cleaving non-coding RNA. Current libraries are a part of a project which aims to identify the major RNA cleavage sites (in mRNA, rRNA and regulatory non-coding RNA) of MazF and MqsR toxins in E. coli. We were also interested in the activity of promoters during toxin overexpression. We used a targeted RNA-sequencing approach that allowed us to map the distinct 5- and 3-ends of toxin-cleaved RNA and also the beginnings of transcripts. We extracted total RNA from cultures where the expression of MazF or MqsR was induced; RNA from log phase culture was used as the control. In addition, RNA extracted from stationary phase culture was used to test for the possible toxin cleavage in natural stress conditions. The cleaved RNA is rapidly recycled making it possible that we miss important cleavage sites due to RNA degradation during the prolonged stationary phase. Therefore, in addition to wt E. coli we also used stationary phase RNA from exoribonuclease deficient strain where RNA cleavage products accumulate. Identification of the 5 ends is based on ligation of RNA adapters to the cellular RNA molecules, which requires 5 -monophosphates. Mapping of the 3 -ends is based on poly(A) tailing of RNA, which requires 3 -OH groups. Transcription initiation, ordinary cellular RNases, and toxin endonucleases all produce different types of RNA ends. These can be enzymatically converted to 5 -phosphates and 3 -OHs, which allows one to separately quantify the RNA ends produced by each of these processes from a single biological sample. Briefly, (i) primary transcripts have 5 -triphosphate ends that can be converted to monophosphate by Tobacco Acid Pyrophosphatase (TAP), and 3 -hydroxyl ends that can be directly polyadenylated by poly(A) polymerase. (ii) Most cellular RNases (excluding RNase I and the toxins) produce 5 -monophosphates and 3 -hydroxyls, which are directly usable for ligation/polyadenylation. (iii) RNase I, MazF, and several other toxins produce 5 -hydroxyl ends that need to be phosphorylated by T4 Polynucleotide Kinase (PNK) prior to ligation, and 2,3 -cyclic phosphates that need to be dephosphorylated and converted to 3 -hydroxyls, also by T4 PNK. In short, for each of our RNA samples three 5 end and two 3 end libraries were made. The PNK treated libraries contain reads mapping specifically to 5- or 3-ends left by toxin (or RNaseI) cleavage and TAP treated libraries have extra reads mapping to beginnings of the transcripts.
2016-11-30 | E-MTAB-4204 | biostudies-arrayexpress