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Decay-initiating endoribonucleolytic cleavage by RNase Y is kept under tight control via sequence preference and sub-cellular localisation

ABSTRACT: Bacteria depend on efficient RNA turnover to rapidly alter gene expression, essentially for responding to changing conditions. Nevertheless, remarkably few details are known about the rate-limiting steps in targeting and decay of RNA. The membrane-anchored endoribonuclease RNase Y is a virulence factor in Gram- positive pathogens. We have obtained a global picture of RNase Y sequence specificity using RNA-seq and the novel transcriptome-wide EMOTE method. Ninety- nine endoribonucleolytic sites produced in vivo were precisely mapped, notably inside six out of seven genes whose half-lives increase the most in an RNase Y deletion mutant, and additionally to three separate transcripts encoding degradation ribonucleases, including RNase Y itself, suggesting a regulatory network. We show that RNase Y is required to initiate the major degradation pathway of a defined sub-set of transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

ORGANISM(S): Staphylococcus aureus

PROVIDER: GSE68811 | GEO | 2015/09/01

SECONDARY ACCESSION(S): PRJNA283825

REPOSITORIES: GEO

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Decay-initiating endoribonucleolytic cleavage by RNase Y is kept under tight control via sequence preference and sub-cellular localisation

Project description:Bacteria depend on efficient RNA turnover to rapidly alter gene expression, essentially for responding to changing conditions. Nevertheless, remarkably few details are known about the rate-limiting steps in targeting and decay of RNA. The membrane-anchored endoribonuclease RNase Y is a virulence factor in Gram- positive pathogens. We have obtained a global picture of RNase Y sequence specificity using RNA-seq and the novel transcriptome-wide EMOTE method. Ninety- nine endoribonucleolytic sites produced in vivo were precisely mapped, notably inside six out of seven genes whose half-lives increase the most in an RNase Y deletion mutant, and additionally to three separate transcripts encoding degradation ribonucleases, including RNase Y itself, suggesting a regulatory network. We show that RNase Y is required to initiate the major degradation pathway of a defined sub-set of transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines. Rnase Y activity in S. aureus is analysed on a genome wide scale under two perspectives: a RNA decay timecourse with mRNA-seq; and exact position of cleavage with an EMOTE assay (Exact Mapping Of Trancripts Ends)

2015-09-01 | E-GEOD-68811 | biostudies-arrayexpress

Decay-Initiating Endoribonucleolytic Cleavage by RNase Y Is Kept under Tight Control via Sequence Preference and Sub-cellular Localisation.

Project description: Not available

| S-EPMC4608709 | biostudies-literature

Expression data from 10-day-old Arabidopsis Col-0 and rns2-2 seedlings

Project description:Housekeeping degradation of normal ribosomal RNA in eukaryotes is completed by the RNase T2 family of ribonucleases. This family of ribonucleases is found through eukaryotes, and the Arabidopsis RNase T2 which degrades ribosomal RNA is RNS2. A null mutant in RNS2, rns2-2, has higher levels of RNA, vacuolar accumulation of rRNA and constitutive autophagy. We used microarray to detail the global changes in gene expression in response to mutation of RNS2 (rns2-2) and identified key biological processes differentially regulated in response to this mutation.

2017-09-15 | GSE81218 | GEO

Transcriptome of Sinorhizobium meliloti RNase mutants

Project description:The ribonucleases (RNases) E and J are essential in Escherichia coli and Bacillus subtilis, respectively. Sinorhizobium meliloti contains both, the rne gene encoding RNase E and the rnj gene encoding RNase J. The transcriptomes of the S. meliloti Rm2011 wild type, and rne and rnj mutants were compared.

2017-01-21 | E-MTAB-5435 | biostudies-arrayexpress

Pleiotropic effects of the rne∆MTS allele on RNA degradation

Project description:Localization of RNase E to the inner membrane in Escherichia coli is well documented, but the functional consequences of this localization are largely unknown. Here we characterize the rne∆MTS strain, which expresses cytoplasmic RNase E (cRNase E). CsrB and CsrC regulatory RNAs are stabilized in the rne∆MTS strain resulting in leaky glycogen expression. There is a small but significant global slowdown in mRNA degradation with no bias considering function or localization of encoded proteins. RNase E is a stable protein, but cRNase E is unstable with a half-life equal to the doubling time of exponentially growing cells. cRNase E instability is compensated by increased synthesis. Co-purification experiments show that cRNase E associates with RhlB, enolase and PNPase to form a cytoplasmic RNA degradosome. Measurements in multiple turnover assays show that there is no difference in Km or kcat between cRNase E and RNase E. In contrast to the global slowdown of mRNA degradation, the inactivation of a ribosome-free lacZ transcript is faster in the rne∆MTS strain. We discuss how the association of RNase E with the inner cytoplasmic membrane is important for carbon storage regulation, degradation of polyribosomal mRNA, protection of ribosome-free transcripts from inactivation and stability of RNase E.

2019-11-25 | GSE118058 | GEO

Unexpected diversity of chloroplast non-coding RNAs as revealed by deep sequencing of the Arabidopsis transcriptome

Project description:Non-coding RNAs (ncRNAs) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly small (18-25 nt) micro- and small interfering RNAs involved in post-transcriptional gene silencing, while prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected, however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein- coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5' end mapping showed that some ncRNAs are transcribed from dedicated promoters, while others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically-transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast.

2011-10-13 | E-MTAB-4478 | biostudies-arrayexpress

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Project description:Ribonucleases are central players in post-transcriptional regulation, a major level of gene expression regulation in all cells. Here, we characterized the 3’-5’ exoribonuclease RNase R from the bacterial pathogen Helicobacter pylori. The "prototypical" Escherichia coli RNase R displays both exoribonuclease and helicase activities, but whether this latter RNA unwinding function is a general feature of bacterial RNase R had not been addressed. We observed that H. pylori HpRNase R protein does not carry the domains responsible for helicase activity and accordingly the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The lack of RNase R helicase domains is widespread among the Campylobacterota, that include Helicobacter and Campylobacter genera, and this loss occurred gradually during their evolution. An in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori was discovered. Purified RhpA facilitates the degradation of double stranded RNA by HpRNase R, showing that this complex is functional. HpRNase R has a minor role in 5S rRNA maturation and few targets in H. pylori, all included in the RhpA regulon. We concluded that during evolution, HpRNase R has co-opted the RhpA helicase to compensate for its lack of helicase activity.

2021-04-08 | PXD023837 | Pride

Attachment of the RNA degradosome to the inner cytoplasmic membrane of Escherichia coli prevents wasteful degradation of rRNA intermediates in ribosome assembly.

Project description:RNase E, an essential endoribonuclease in Escherichia coli, is the key component of the multienzyme RNA degradosome, which is localized to the inner cytoplasmic membrane. Until now, the reason for membrane localization of RNase E was not known. We have analyzed ribosome assembly in the rneΔMTS strain, which expresses a cytoplasmic variant of RNase E (cRNase E) resulting in a cytoplasmic RNA degradosome. In this mutant strain, there is a mild slowdown in the rates of growth and mRNA degradation. Here we document the striking accumulation of intermediates in ribosome assembly in the rneΔMTS strain in which precursors of 16S and 23S rRNA are cleaved by cRNase E. In vitro, we show that ribosomes partially unfolded in low ionic strength buffer are cleaved by RNase E. Mapping of in vivo and in vitro cleavage sites shows that they overlap and that their consensus sequence matches previously mapped RNase E cleavage sites. In vivo, fragments of 16S and 23S rRNA as well as a precursor of 5S rRNA are degraded in a pathway involving polyadenylation and 3’ exonucleases. Since the pathway for rRNA degradation is the same as the pathway for mRNA degradation, the slowdown of mRNA degradation in the rneΔMTS strain could be due to competition by rRNA degradation. Our results strongly suggest that the RNA degradosome participates in the quality control of ribosome assembly and that localization on the inner cytoplasmic membrane protects newly synthesized intermediates from wasteful degradation. Ribosome synthesis is costly. Since growth rate correlates with ribosome synthesis rate, slow growth rate of the rneΔMTS strain is due to the degradation of a proportion of newly synthesized ribosomes. Avoiding wasteful degradation of intermediates in ribosome assembly likely explains why localization of RNase E homologues to the inner cytoplasmic membrane is conserved throughout the γ-Proteobacteria.

2023-01-16 | PXD038317 | Pride

RNase R is associated in a functional complex with the RhpA DEAD-box RNA helicase in Helicobacter pylori

Project description:Ribonucleases (RNases) are central actors in post-transcriptional regulation, a major level of regulation of gene expression in all cells. This control plays an important role in the bacterial pathogen Helicobacter pylori, although only the function of RNase J was characterized so far. Here, we studied the RNase R enzyme from H. pylori, a 3’-5’ exoribonuclease whose ortholog in Escherichia coli was reported to display, in addition, helicase activity and to be able to hydrolyze RNA substrates with double stranded structures. We observed that HpRNase R protein does not carry the domains responsible for helicase activity in E. coli and accordingly that the purified protein is unable to degrade in vitro RNA molecules with secondary structures. The RNase R helicase domain loss is widespread among the Campylobacterota and occurred gradually during their evolution. Furthermore, an in vivo interaction between HpRNase R and RhpA, the sole DEAD-box RNA helicase of H. pylori, was discovered. Phylogenomics suggests that this interaction might occur in other bacteria of the phylum Campylobacterota. Purified RhpA facilitates the degradation of double stranded RNA substrates by HpRNase R, showing that this complex is functional. HpRNase R has a minor role of in 5S rRNA maturation and, as shown by RNA-Seq, few targets in H. pylori all of them being included in the RhpA regulon. In conclusion, we describe a new type of RNase R that lacks some of the features that were considered as hallmarks of RNase R proteins, but that has co-opted another RNA helicase, which we hypothesize helps it accomplish some of its functions in vivo.

2021-06-19 | E-MTAB-10045 | biostudies-arrayexpress

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artifacts when imaging RNA:DNA hybrids

Project description:We showed that the majority of the signal observed in S9.6 immunofluorescence microscopy images of fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining is quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification that the enzyme was active in situ. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

2021-03-17 | GSE141833 | GEO

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