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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

T-box riboswitches cleaved by RNase Y.(A) Layout of the valS T-box, indicating Transcription Start Site (TSS), RNase Y cleavage site (Y-cleavage), the highly conserved T-box motif that binds to the 3' CCA of uncharged tRNAs (Anti-CCA), and the predicted T-box riboswitch transcriptional terminator (TB-Term.). The thick line indicate the terminated T-box RNA, and the dotted thin line indicate the full-length transcript. The approximate extent of the fragment that appears in the J1AGA mutant is shown with a thin line. The probe used for the Northern blot in panel B, is indicated with a thick grey bar. (B) Northern blot showing the terminated glyS T-box riboswitch (TB). A fragment downstream of the RNase Y cleavage site accumulates in the RNase J mutant (Y). Additionally, a sub-fragment of about 125 nt is detectable in the ΔY and Y367AA strains (asterisk). Measured sizes of the detected fragments are indicated to the right. Relative intensities are indicated above each band. Loading was normalised to 5S rRNA (shown separately underneath), and the intensity of the band corresponding to the full-length T-box in WT lane set to 1. (C and D) The leuS T-box, with indications like panel A and B. (E and F). The serS T-box, with indications like panel A and B. (G and H) The glyS T-box, with indications like panel A and B.
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pgen.1005577.g003: T-box riboswitches cleaved by RNase Y.(A) Layout of the valS T-box, indicating Transcription Start Site (TSS), RNase Y cleavage site (Y-cleavage), the highly conserved T-box motif that binds to the 3' CCA of uncharged tRNAs (Anti-CCA), and the predicted T-box riboswitch transcriptional terminator (TB-Term.). The thick line indicate the terminated T-box RNA, and the dotted thin line indicate the full-length transcript. The approximate extent of the fragment that appears in the J1AGA mutant is shown with a thin line. The probe used for the Northern blot in panel B, is indicated with a thick grey bar. (B) Northern blot showing the terminated glyS T-box riboswitch (TB). A fragment downstream of the RNase Y cleavage site accumulates in the RNase J mutant (Y). Additionally, a sub-fragment of about 125 nt is detectable in the ΔY and Y367AA strains (asterisk). Measured sizes of the detected fragments are indicated to the right. Relative intensities are indicated above each band. Loading was normalised to 5S rRNA (shown separately underneath), and the intensity of the band corresponding to the full-length T-box in WT lane set to 1. (C and D) The leuS T-box, with indications like panel A and B. (E and F). The serS T-box, with indications like panel A and B. (G and H) The glyS T-box, with indications like panel A and B.

Mentions: From the list of putative RNase Y cleavage sites identified by EMOTE, it was striking that seven sites were located immediately upstream of aminoacyl-tRNA synthetases. This group of genes are often regulated by T-box riboswitches, which sense the level of tRNA molecules charged with amino acids, and terminate transcription prematurely if the level is already sufficient [31]. Four predicted T-box riboswitches were examined by Northern blotting: TB-valS, TB-leuS, TB-serS and TB-glyS. The prematurely terminated 5'-UTR was observed for all four T-boxes, and the accumulation observed by RNA-seq was confirmed for TB-glyS, TB-serS, and TB-valS (Fig 3). No degradation intermediates were observed to accumulate significantly in neither WT nor RNase Y mutant strains, presumably because degradation initiated by RNase Y is the rate-limiting step. Therefore, in order to observe the cleavage site identified by EMOTE, a mutant with the highly efficient 5' to 3' exoribonuclease J1 inactivated (Strain J1AGA) was used to prevent further processing of the downstream cleavage products generated by RNase Y. This approach revealed RNA fragments corresponding to the distance between the RNase Y cleavage and the riboswitch-induced transcription termination site for valS and leuS T-boxes (Fig 3B and 3D). In contrast, the downstream fragments that appear for the glyS and serS T-boxes are shorter than the distance between the RNase Y cleavage site and the T-box transcriptional terminator. The glyS T-box was therefore examined further, by using three additional probes to cover the entire 204 nt RNA. The two probes hybridising upstream of the RNase Y cleavage site were unable to detect the fragment that accumulates in J1AGA, demonstrating that it is indeed a result of cleavage at position +111, and is probably slightly shortened by 3' to 5' exonucleases until one of the many T-box secondary structures prevent further 3' processing (S7 Fig).


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)

T-box riboswitches cleaved by RNase Y.(A) Layout of the valS T-box, indicating Transcription Start Site (TSS), RNase Y cleavage site (Y-cleavage), the highly conserved T-box motif that binds to the 3' CCA of uncharged tRNAs (Anti-CCA), and the predicted T-box riboswitch transcriptional terminator (TB-Term.). The thick line indicate the terminated T-box RNA, and the dotted thin line indicate the full-length transcript. The approximate extent of the fragment that appears in the J1AGA mutant is shown with a thin line. The probe used for the Northern blot in panel B, is indicated with a thick grey bar. (B) Northern blot showing the terminated glyS T-box riboswitch (TB). A fragment downstream of the RNase Y cleavage site accumulates in the RNase J mutant (Y). Additionally, a sub-fragment of about 125 nt is detectable in the ΔY and Y367AA strains (asterisk). Measured sizes of the detected fragments are indicated to the right. Relative intensities are indicated above each band. Loading was normalised to 5S rRNA (shown separately underneath), and the intensity of the band corresponding to the full-length T-box in WT lane set to 1. (C and D) The leuS T-box, with indications like panel A and B. (E and F). The serS T-box, with indications like panel A and B. (G and H) The glyS T-box, with indications like panel A and B.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1005577.g003: T-box riboswitches cleaved by RNase Y.(A) Layout of the valS T-box, indicating Transcription Start Site (TSS), RNase Y cleavage site (Y-cleavage), the highly conserved T-box motif that binds to the 3' CCA of uncharged tRNAs (Anti-CCA), and the predicted T-box riboswitch transcriptional terminator (TB-Term.). The thick line indicate the terminated T-box RNA, and the dotted thin line indicate the full-length transcript. The approximate extent of the fragment that appears in the J1AGA mutant is shown with a thin line. The probe used for the Northern blot in panel B, is indicated with a thick grey bar. (B) Northern blot showing the terminated glyS T-box riboswitch (TB). A fragment downstream of the RNase Y cleavage site accumulates in the RNase J mutant (Y). Additionally, a sub-fragment of about 125 nt is detectable in the ΔY and Y367AA strains (asterisk). Measured sizes of the detected fragments are indicated to the right. Relative intensities are indicated above each band. Loading was normalised to 5S rRNA (shown separately underneath), and the intensity of the band corresponding to the full-length T-box in WT lane set to 1. (C and D) The leuS T-box, with indications like panel A and B. (E and F). The serS T-box, with indications like panel A and B. (G and H) The glyS T-box, with indications like panel A and B.
Mentions: From the list of putative RNase Y cleavage sites identified by EMOTE, it was striking that seven sites were located immediately upstream of aminoacyl-tRNA synthetases. This group of genes are often regulated by T-box riboswitches, which sense the level of tRNA molecules charged with amino acids, and terminate transcription prematurely if the level is already sufficient [31]. Four predicted T-box riboswitches were examined by Northern blotting: TB-valS, TB-leuS, TB-serS and TB-glyS. The prematurely terminated 5'-UTR was observed for all four T-boxes, and the accumulation observed by RNA-seq was confirmed for TB-glyS, TB-serS, and TB-valS (Fig 3). No degradation intermediates were observed to accumulate significantly in neither WT nor RNase Y mutant strains, presumably because degradation initiated by RNase Y is the rate-limiting step. Therefore, in order to observe the cleavage site identified by EMOTE, a mutant with the highly efficient 5' to 3' exoribonuclease J1 inactivated (Strain J1AGA) was used to prevent further processing of the downstream cleavage products generated by RNase Y. This approach revealed RNA fragments corresponding to the distance between the RNase Y cleavage and the riboswitch-induced transcription termination site for valS and leuS T-boxes (Fig 3B and 3D). In contrast, the downstream fragments that appear for the glyS and serS T-boxes are shorter than the distance between the RNase Y cleavage site and the T-box transcriptional terminator. The glyS T-box was therefore examined further, by using three additional probes to cover the entire 204 nt RNA. The two probes hybridising upstream of the RNase Y cleavage site were unable to detect the fragment that accumulates in J1AGA, demonstrating that it is indeed a result of cleavage at position +111, and is probably slightly shortened by 3' to 5' exonucleases until one of the many T-box secondary structures prevent further 3' processing (S7 Fig).

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