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

Khemici V, Prados J, Linder P, Redder P - PLoS Genet. (2015)

Bottom Line: We have obtained a global picture of Staphylococcus aureus 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 in 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 about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Switzerland.

ABSTRACT
Bacteria depend on efficient RNA turnover, both during homeostasis and when rapidly altering gene expression in response to changes. 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 Staphylococcus aureus 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 in 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 about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

No MeSH data available.


Related in: MedlinePlus

RNase Y cleavage sites fall within or near the genes encoding components of the putative degradosome, as defined by [17], and show sequence preference for guanosines.Predicted transcription start sites (TSS), detected RNase Y cleavage sites (Y-cleav.) and predicted transcriptional terminators (TransTerm) are indicated. Thin dotted lines show the sequence amplified by RT-PCR to demonstrate that RNase Y cleavage sites and ORFs are potentially on the same RNA molecules. (A) SA0941-rnjA operon, encoding a hypothetical protein and RNase J1. (B) rnjB gene encoding RNase J2 (pnpA encodes PNPase). (C) pfkA-pykA operon, encoding phosphofructokinase and pyruvate kinase. (D) The RNase Y gene (cvfA) with the putative RNase Y cleavage site at position +24. (E) RNase Y cleaves preferentially after a purine. 25 nucleotides on each side of the 99 RNase Y sites (see S2 Table) were extracted from the S. aureus N315 genome, and a frequency plot was generated. The RNase Y cleavage site (thick vertical dotted line), and the position detected by EMOTE (Y+1) are indicated. The preference for a guanosine residue immediately prior to the RNase Y cleavage sites is evident. Additionally, there is an enrichment of A's at position Y–6 and of pyrimidines at position Y+5. Finally, it appears that A's or U's are favoured in the region from 11 nt upstream to 7 nt downstream of the cleavage site, however this could also be due to the over-all nucleotide composition of S. aureus, which has 33% G+C (thin horizontal dotted line).
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pgen.1005577.g002: RNase Y cleavage sites fall within or near the genes encoding components of the putative degradosome, as defined by [17], and show sequence preference for guanosines.Predicted transcription start sites (TSS), detected RNase Y cleavage sites (Y-cleav.) and predicted transcriptional terminators (TransTerm) are indicated. Thin dotted lines show the sequence amplified by RT-PCR to demonstrate that RNase Y cleavage sites and ORFs are potentially on the same RNA molecules. (A) SA0941-rnjA operon, encoding a hypothetical protein and RNase J1. (B) rnjB gene encoding RNase J2 (pnpA encodes PNPase). (C) pfkA-pykA operon, encoding phosphofructokinase and pyruvate kinase. (D) The RNase Y gene (cvfA) with the putative RNase Y cleavage site at position +24. (E) RNase Y cleaves preferentially after a purine. 25 nucleotides on each side of the 99 RNase Y sites (see S2 Table) were extracted from the S. aureus N315 genome, and a frequency plot was generated. The RNase Y cleavage site (thick vertical dotted line), and the position detected by EMOTE (Y+1) are indicated. The preference for a guanosine residue immediately prior to the RNase Y cleavage sites is evident. Additionally, there is an enrichment of A's at position Y–6 and of pyrimidines at position Y+5. Finally, it appears that A's or U's are favoured in the region from 11 nt upstream to 7 nt downstream of the cleavage site, however this could also be due to the over-all nucleotide composition of S. aureus, which has 33% G+C (thin horizontal dotted line).

Mentions: One identified cleavage site corresponded to the previously demonstrated RNase Y cleavage in the sae operon [8]. A number of the identified RNase Y cleavage sites fell within nine ORFs that were stabilised in our transcriptome wide RNA stability assay, showing that these coding RNAs are direct targets of RNase Y, which performs the cleavage that initiates degradation (Table 2, Fig 1). However, a decay-initiating cleavage does not need to be within an ORF, but can in principle be anywhere on the RNA. Therefore the search was extended 200 nucleotides in either direction from the ORFs, which identified an additional ten stabilised ORFs with associated RNase Y cleavage sites. To ensure that these ORFs are potentially on the same RNA molecules as the detected RNase Y cleavage, reads were identified in the RNA-seq data that span both the cleavage site and the ORF, with >50 reads considered significant. An RT-PCR assay was used to connect those sites/ORFs that either have too few RNA-seq reads or are further from the ORF than the length of the Illumina reads (50 nt). Combined, the RT-PCR reactions and the RNA-seq connected 37 cleavage sites with corresponding ORFs (S4 Fig), including all ORFs that are significantly stabilised in the RNase Y mutant. Thus a total of 19 stabilised ORFs (20%) could be linked to one or more of the 99 identified RNase Y cleavage sites. Moreover, RNase Y cleavage sites were identified in or near RNase J1 (SA0941-rnjA operon), RNase J2 (rnjB) and phosphofructokinase (pfkA-pykA operon), which have all been linked to the RNA decay machinery (Fig 2A–2C) [17]. Finally, because the RNase Y-encoding cvfA gene is not expressed in the ΔY strain it is inherently excluded by the criteria used to identify RNase Y cleavage sites, however, when EMOTE data from the Y367AA strain (which does express cvfA) is compared to WT data, then a potential RNase Y cleavage site inside its own transcript is revealed (Figs 2D and S4; Table 2).


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

Khemici V, Prados J, Linder P, Redder P - PLoS Genet. (2015)

RNase Y cleavage sites fall within or near the genes encoding components of the putative degradosome, as defined by [17], and show sequence preference for guanosines.Predicted transcription start sites (TSS), detected RNase Y cleavage sites (Y-cleav.) and predicted transcriptional terminators (TransTerm) are indicated. Thin dotted lines show the sequence amplified by RT-PCR to demonstrate that RNase Y cleavage sites and ORFs are potentially on the same RNA molecules. (A) SA0941-rnjA operon, encoding a hypothetical protein and RNase J1. (B) rnjB gene encoding RNase J2 (pnpA encodes PNPase). (C) pfkA-pykA operon, encoding phosphofructokinase and pyruvate kinase. (D) The RNase Y gene (cvfA) with the putative RNase Y cleavage site at position +24. (E) RNase Y cleaves preferentially after a purine. 25 nucleotides on each side of the 99 RNase Y sites (see S2 Table) were extracted from the S. aureus N315 genome, and a frequency plot was generated. The RNase Y cleavage site (thick vertical dotted line), and the position detected by EMOTE (Y+1) are indicated. The preference for a guanosine residue immediately prior to the RNase Y cleavage sites is evident. Additionally, there is an enrichment of A's at position Y–6 and of pyrimidines at position Y+5. Finally, it appears that A's or U's are favoured in the region from 11 nt upstream to 7 nt downstream of the cleavage site, however this could also be due to the over-all nucleotide composition of S. aureus, which has 33% G+C (thin horizontal dotted line).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4608709&req=5

pgen.1005577.g002: RNase Y cleavage sites fall within or near the genes encoding components of the putative degradosome, as defined by [17], and show sequence preference for guanosines.Predicted transcription start sites (TSS), detected RNase Y cleavage sites (Y-cleav.) and predicted transcriptional terminators (TransTerm) are indicated. Thin dotted lines show the sequence amplified by RT-PCR to demonstrate that RNase Y cleavage sites and ORFs are potentially on the same RNA molecules. (A) SA0941-rnjA operon, encoding a hypothetical protein and RNase J1. (B) rnjB gene encoding RNase J2 (pnpA encodes PNPase). (C) pfkA-pykA operon, encoding phosphofructokinase and pyruvate kinase. (D) The RNase Y gene (cvfA) with the putative RNase Y cleavage site at position +24. (E) RNase Y cleaves preferentially after a purine. 25 nucleotides on each side of the 99 RNase Y sites (see S2 Table) were extracted from the S. aureus N315 genome, and a frequency plot was generated. The RNase Y cleavage site (thick vertical dotted line), and the position detected by EMOTE (Y+1) are indicated. The preference for a guanosine residue immediately prior to the RNase Y cleavage sites is evident. Additionally, there is an enrichment of A's at position Y–6 and of pyrimidines at position Y+5. Finally, it appears that A's or U's are favoured in the region from 11 nt upstream to 7 nt downstream of the cleavage site, however this could also be due to the over-all nucleotide composition of S. aureus, which has 33% G+C (thin horizontal dotted line).
Mentions: One identified cleavage site corresponded to the previously demonstrated RNase Y cleavage in the sae operon [8]. A number of the identified RNase Y cleavage sites fell within nine ORFs that were stabilised in our transcriptome wide RNA stability assay, showing that these coding RNAs are direct targets of RNase Y, which performs the cleavage that initiates degradation (Table 2, Fig 1). However, a decay-initiating cleavage does not need to be within an ORF, but can in principle be anywhere on the RNA. Therefore the search was extended 200 nucleotides in either direction from the ORFs, which identified an additional ten stabilised ORFs with associated RNase Y cleavage sites. To ensure that these ORFs are potentially on the same RNA molecules as the detected RNase Y cleavage, reads were identified in the RNA-seq data that span both the cleavage site and the ORF, with >50 reads considered significant. An RT-PCR assay was used to connect those sites/ORFs that either have too few RNA-seq reads or are further from the ORF than the length of the Illumina reads (50 nt). Combined, the RT-PCR reactions and the RNA-seq connected 37 cleavage sites with corresponding ORFs (S4 Fig), including all ORFs that are significantly stabilised in the RNase Y mutant. Thus a total of 19 stabilised ORFs (20%) could be linked to one or more of the 99 identified RNase Y cleavage sites. Moreover, RNase Y cleavage sites were identified in or near RNase J1 (SA0941-rnjA operon), RNase J2 (rnjB) and phosphofructokinase (pfkA-pykA operon), which have all been linked to the RNA decay machinery (Fig 2A–2C) [17]. Finally, because the RNase Y-encoding cvfA gene is not expressed in the ΔY strain it is inherently excluded by the criteria used to identify RNase Y cleavage sites, however, when EMOTE data from the Y367AA strain (which does express cvfA) is compared to WT data, then a potential RNase Y cleavage site inside its own transcript is revealed (Figs 2D and S4; Table 2).

Bottom Line: We have obtained a global picture of Staphylococcus aureus 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 in 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 about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Molecular Medicine, Faculty of Medicine, University of Geneva, Switzerland.

ABSTRACT
Bacteria depend on efficient RNA turnover, both during homeostasis and when rapidly altering gene expression in response to changes. 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 Staphylococcus aureus 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 in 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 about a hundred transcripts that are inaccessible to other ribonucleases, but is prevented from promiscuous activity by membrane confinement and sequence preference for guanosines.

No MeSH data available.


Related in: MedlinePlus