Bacterial RNase III cleaves long dsRNA at preferred sites
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ABSTRACT:
PROVIDER: PRJNA491502 | ENA |
REPOSITORIES: ENA
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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:RNase III is a ribonucleases that recognizes and cleaves double-stranded RNA. RNase III has been known be involved in rRNA processing, but has many additional roles controlling both expression and RNA turnover of specific messages. Many organisms have just one RNase III while some have both a full length RNase III and a mini-III that lacks the double-stranded RNA binding domain. The cyanobacteria Synechococcus sp. PCC 7002 has three homologs of RNase III that are unessential even when deleted in combination. We were interested what coding regions these RNase III enzymes were influencing and if they had redundant or distinct specificities. To address these questions we collected samples for RNA-sequencing from WT, the single, double, and triple RNase III mutants in triplicate. Approximately 20% of genes were differentially expressed in various mutants with some operons and regulons showing complex changes in expression levels between mutants. We describe the role of two RNase III’s in 23S rRNA maturation, and show how the third is involved in copy number regulation of one of the six plasmids (pAQ3). Purified enzymes were capable of cleaving some E. coli RNase III target sequences, highlighting the remarkably conserved substrate specificity between organisms yet complex regulation of gene expression.
2018-01-16 | GSE99279 | GEO
Project description:Our results demonstrate that RNase G controls expression levels of H-NS encoded by hns, strongly associated with the pathogenicity of S. Typhimurium. The hns mRNA abundance is mediated by RNase G, which cleaves the 5’-UTR of hns mRNA. In the upstream pathway, the induced expression of RNase G in host environment condition is attributable to reduced RNase III cleavage activity on rng mRNA. Our findings suggest the link between Salmonella pathogenicity and RNase III-RNase G pathway, underlining the importance of posttranscriptional regulation of H-NS in the downstream pathway, which controls Salmonella pathogenicity island-1 type III secretion system for the survival and virulence of S. Typhimurium in host cell.
2021-01-08 | PXD011090 | Pride
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:RNAse III is an evolutionarily conserved family of endoribonuclease that cleaves dsRNA structures. Here we studied RNAse III homolog Pac1 In fission yeast to shed light on underappreciated roles of Drosha. We found that Pac1 co-transcriptional cleavage of nascent hairpin RNA structures trigger transcriptional termination by creating an entry point for “torpedo” exonucleases – that is, exonuclease that degrade the nascent RNA until they bump into the transcribing polymerases. As such termination pathway decouple termination from pre-mRNA polyadenylation, the nascent transcript are destabilized upon Pac1 cleavage, therefore expending the paradigm of post-transcriptional regulation of gene expression.
2021-02-19 | GSE167041 | GEO
Project description:RNAse III is an evolutionarily conserved family of endoribonuclease that cleaves dsRNA structures. Here we studied RNAse III homolog Pac1 In fission yeast to shed light on underappreciated roles of Drosha. We found that Pac1 co-transcriptional cleavage of nascent hairpin RNA structures trigger transcriptional termination by creating an entry point for “torpedo” exonucleases – that is, exonuclease that degrade the nascent RNA until they bump into the transcribing polymerases. As such termination pathway decouple termination from pre-mRNA polyadenylation, the nascent transcript are destabilized upon Pac1 cleavage, therefore expending the paradigm of post-transcriptional regulation of gene expression.
2021-02-19 | GSE167040 | GEO
Project description:RNase III is an important and highly conserved endoribonuclease known to impact rRNA, mRNA and ncRNA abundances by RNA processing. In this study we analyzed the effects of an inactivation of RNase III (inactivated through substitution of two strictly conserved amino acids within the active enzyme center) on the transcriptome of the facultative phototrophic model organism Rhodobacter sphaeroides.
2024-02-19 | GSE236804 | GEO
Project description:The Corynebacterium glutamicum R cgR_1959 gene encodes an endoribonuclease of the RNase III family. Deletion mutant of cgR_1959 (Δrnc mutant) showed an elongated cell shape, and presence of several lines on the cell surface, indicating a required of RNase III for maintaining normal cell morphology in C. glutamicum. The level of mraZ mRNA was increased, whereas cgR_1596 mRNA encoding a putative cell wall hydrolase and ftsEX mRNA were decreased in the Δrnc mutant. The half-life of mraZ mRNA was significantly prolonged in the Δrnc and the Δpnp mutant strains. This indicated that the degradation of mraZ mRNA was performed by RNase III and the 3′-to-5′ exoribonuclease, PNPase. Northern hybridization and primer extension analysis revealed that the cleavage site for mraZ mRNA by RNase III is in the coding region. Overproduction of MraZ resulted in an elongated cell shape. The expression of ftsEX decreased while that of cgR_1596 unchanged in an MraZ-overexpressing strain. An electrophoretic mobility shift assay and a transcriptional reporter assay indicate that MraZ is a transcriptional repressor of ftsEX in C. glutamicum. These results indicate that RNase III is required for efficient expression of MraZ-dependent ftsEX and MraZ-independent cgR_1596.
2016-01-17 | GSE75080 | GEO
Project description:Bordetella pertussis has been shown to encode regulatory RNAs, yet the post-transcriptional regulatory circuits on which they act remain to be fully elucidated. We generated mutants lacking the endonucleases RNase III and RNase E and assessed their individual impact on the B. pertussis transcriptome. RNA-Seq analysis showed differential expression of ~25% of the B. pertussis transcriptome in each mutant with only 28% overlap between data sets. Both endonucleases exhibited substantial impact on genes involved in amino acid uptake e.g. ABC transporters, and in virulence e.g. the type III secretion system, and the autotransporters vag8, tcfA and brkA. Interestingly, mutations in RNase III and RNase E drove the stability of many transcripts, including those involved in virulence, in opposite directions; a result that was validated by qPCR and immunoblot for tcfA and brkA. Of note, whereas similar mutations to RNase E in E. coli have subtle effects on transcript stability, a striking >20-fold reduction in four gene transcripts, including tcfA and vag8, was observed in B. pertussis. We further compared our data set to the regulon controlled by the RNA chaperone Hfq to identify B. pertussis loci influenced by regulatory RNAs. This analysis identified ~120 genes and 19 operons potentially regulated at the post-transcriptional level. Thus, our findings revealed how changes in RNase III- and RNase E-mediated RNA turnover influence pathways associated with virulence and cellular homeostasis. Moreover, we highlighted loci potentially influenced by regulatory RNAs, providing insights into the post-transcriptional regulatory networks involved in fine tuning B. pertussis gene expression.
2021-08-11 | GSE164312 | GEO