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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

rpsB and tsf are co-transcribed via partial read-through of a transcriptional terminator, and both transcripts are stabilised in ΔY.(A) Overview of the rpsB-tsf locus, with the various predicted transcription start sites (TSSs) indicated. The five probes used for Northern blotting are shown in grey, and the location of the fragment detected with probe C in the J1AGA mutant is highlighted by a thick black arrow. Thin arrows indicate open reading frames, including the short unannotated ORF X. RNase Y cleavage sites are marked in bold, and the question mark indicates the frequent read-through of the transcriptional terminator located inside ORF X. Thin dotted arrows indicate the amplification by RT-PCR (see S4 Fig). (B) Predicted hairpin structure of the transcriptional terminator at position +947. Panels C to F show the Northern blot membrane probed with rpsB-C, rpsB-D, tsf-E and stained with methylene blue as loading control. The read-through transcript (*), the rpsB transcript (#) and the tsf transcript (§) are indicated. Panels G to I show Northern blots optimised for short fragments (8% acryl-amide/urea gel) and probed with rpsB-C and rpsB-D, as well as a 5S-rRNA probe as loading control. In panel G, note the ~75 nt RNA that accumulate in the J1AGA mutant (thick black arrow) and a minor band just above (thin dotted arrow).
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pgen.1005577.g004: rpsB and tsf are co-transcribed via partial read-through of a transcriptional terminator, and both transcripts are stabilised in ΔY.(A) Overview of the rpsB-tsf locus, with the various predicted transcription start sites (TSSs) indicated. The five probes used for Northern blotting are shown in grey, and the location of the fragment detected with probe C in the J1AGA mutant is highlighted by a thick black arrow. Thin arrows indicate open reading frames, including the short unannotated ORF X. RNase Y cleavage sites are marked in bold, and the question mark indicates the frequent read-through of the transcriptional terminator located inside ORF X. Thin dotted arrows indicate the amplification by RT-PCR (see S4 Fig). (B) Predicted hairpin structure of the transcriptional terminator at position +947. Panels C to F show the Northern blot membrane probed with rpsB-C, rpsB-D, tsf-E and stained with methylene blue as loading control. The read-through transcript (*), the rpsB transcript (#) and the tsf transcript (§) are indicated. Panels G to I show Northern blots optimised for short fragments (8% acryl-amide/urea gel) and probed with rpsB-C and rpsB-D, as well as a 5S-rRNA probe as loading control. In panel G, note the ~75 nt RNA that accumulate in the J1AGA mutant (thick black arrow) and a minor band just above (thin dotted arrow).

Mentions: The loss of RNase Y activity generates a dramatic effect for the rpsB and tsf ORFs (encoding ribosomal protein S2 and elongation factor TS, respectively), both in terms of half-life and steady-state levels (Fig 1, Tables 2 and S1). A predicted rho-independent transcriptional terminator can be identified between these two genes (Fig 4B and position +947 in Fig 4A; [32]), but previous studies, based on both microarray and RNA-seq data, suggest that they might be co-transcribed [32,33]. The identification, by EMOTE, of an RNase Y cleavage site inside the rpsB ORF (Fig 4A, position +865), suggests that RNase Y can initiate decay of the rpsB transcript, and thereby influence the stability, and consequently the steady-state level. However, no RNase Y sites were detected in the tsf transcript, and we therefore examined whether rpsB and tsf are indeed in an operon (i.e. whether there is read-through of the transcriptional terminator), and whether the steady-state levels of such a read-through transcript are affected by the deletion of RNase Y. Northern blotting with probe rpsB-C, which anneals immediately upstream of the transcriptional terminator (Fig 4A and 4C) revealed a major transcript of ~900 nt (corresponding to termination at the terminator) and a slightly weaker signal at ~2000 nt (corresponding to read-through of the terminator). As expected from the RNA-seq, both of these transcripts were massively accumulated in the ΔY strain (Fig 4C), indicating that the stabilisation of rpsB and tsf observed in the rifampicin assay (Fig 1) is the cause, although it cannot be ruled out that the ΔY mutant exhibits increased rpsB promoter activity, which would also contribute to the increase in steady state level. A probe annealing inside the tsf ORF (Fig 4E), also gave rise to two bands in the WT strain: a ~2000 nt transcript, corresponding to the read-through from the rpsB promoter, and a ~1000 nt RNA which would correspond to a transcript from the predicted tsf promoter (Fig 4A, position +1041). In the ΔY strain, the signal for the ~2000 nt band is much stronger than for the ~1000 nt band, indicating that whatever affects the tsf-encoding RNA must happen upstream of tsf transcription start site. Probe rpsB-D hybridises between the transcriptional terminator and the tsf transcription start site, and detected neither the ~900 nt band from probe rpsB-C nor the ~1000 nt band from probe tsf-E, verifying that these transcripts do not overlap (Fig 4D).


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)

rpsB and tsf are co-transcribed via partial read-through of a transcriptional terminator, and both transcripts are stabilised in ΔY.(A) Overview of the rpsB-tsf locus, with the various predicted transcription start sites (TSSs) indicated. The five probes used for Northern blotting are shown in grey, and the location of the fragment detected with probe C in the J1AGA mutant is highlighted by a thick black arrow. Thin arrows indicate open reading frames, including the short unannotated ORF X. RNase Y cleavage sites are marked in bold, and the question mark indicates the frequent read-through of the transcriptional terminator located inside ORF X. Thin dotted arrows indicate the amplification by RT-PCR (see S4 Fig). (B) Predicted hairpin structure of the transcriptional terminator at position +947. Panels C to F show the Northern blot membrane probed with rpsB-C, rpsB-D, tsf-E and stained with methylene blue as loading control. The read-through transcript (*), the rpsB transcript (#) and the tsf transcript (§) are indicated. Panels G to I show Northern blots optimised for short fragments (8% acryl-amide/urea gel) and probed with rpsB-C and rpsB-D, as well as a 5S-rRNA probe as loading control. In panel G, note the ~75 nt RNA that accumulate in the J1AGA mutant (thick black arrow) and a minor band just above (thin dotted arrow).
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Related In: Results  -  Collection

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pgen.1005577.g004: rpsB and tsf are co-transcribed via partial read-through of a transcriptional terminator, and both transcripts are stabilised in ΔY.(A) Overview of the rpsB-tsf locus, with the various predicted transcription start sites (TSSs) indicated. The five probes used for Northern blotting are shown in grey, and the location of the fragment detected with probe C in the J1AGA mutant is highlighted by a thick black arrow. Thin arrows indicate open reading frames, including the short unannotated ORF X. RNase Y cleavage sites are marked in bold, and the question mark indicates the frequent read-through of the transcriptional terminator located inside ORF X. Thin dotted arrows indicate the amplification by RT-PCR (see S4 Fig). (B) Predicted hairpin structure of the transcriptional terminator at position +947. Panels C to F show the Northern blot membrane probed with rpsB-C, rpsB-D, tsf-E and stained with methylene blue as loading control. The read-through transcript (*), the rpsB transcript (#) and the tsf transcript (§) are indicated. Panels G to I show Northern blots optimised for short fragments (8% acryl-amide/urea gel) and probed with rpsB-C and rpsB-D, as well as a 5S-rRNA probe as loading control. In panel G, note the ~75 nt RNA that accumulate in the J1AGA mutant (thick black arrow) and a minor band just above (thin dotted arrow).
Mentions: The loss of RNase Y activity generates a dramatic effect for the rpsB and tsf ORFs (encoding ribosomal protein S2 and elongation factor TS, respectively), both in terms of half-life and steady-state levels (Fig 1, Tables 2 and S1). A predicted rho-independent transcriptional terminator can be identified between these two genes (Fig 4B and position +947 in Fig 4A; [32]), but previous studies, based on both microarray and RNA-seq data, suggest that they might be co-transcribed [32,33]. The identification, by EMOTE, of an RNase Y cleavage site inside the rpsB ORF (Fig 4A, position +865), suggests that RNase Y can initiate decay of the rpsB transcript, and thereby influence the stability, and consequently the steady-state level. However, no RNase Y sites were detected in the tsf transcript, and we therefore examined whether rpsB and tsf are indeed in an operon (i.e. whether there is read-through of the transcriptional terminator), and whether the steady-state levels of such a read-through transcript are affected by the deletion of RNase Y. Northern blotting with probe rpsB-C, which anneals immediately upstream of the transcriptional terminator (Fig 4A and 4C) revealed a major transcript of ~900 nt (corresponding to termination at the terminator) and a slightly weaker signal at ~2000 nt (corresponding to read-through of the terminator). As expected from the RNA-seq, both of these transcripts were massively accumulated in the ΔY strain (Fig 4C), indicating that the stabilisation of rpsB and tsf observed in the rifampicin assay (Fig 1) is the cause, although it cannot be ruled out that the ΔY mutant exhibits increased rpsB promoter activity, which would also contribute to the increase in steady state level. A probe annealing inside the tsf ORF (Fig 4E), also gave rise to two bands in the WT strain: a ~2000 nt transcript, corresponding to the read-through from the rpsB promoter, and a ~1000 nt RNA which would correspond to a transcript from the predicted tsf promoter (Fig 4A, position +1041). In the ΔY strain, the signal for the ~2000 nt band is much stronger than for the ~1000 nt band, indicating that whatever affects the tsf-encoding RNA must happen upstream of tsf transcription start site. Probe rpsB-D hybridises between the transcriptional terminator and the tsf transcription start site, and detected neither the ~900 nt band from probe rpsB-C nor the ~1000 nt band from probe tsf-E, verifying that these transcripts do not overlap (Fig 4D).

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