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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

Removal of the membrane anchor enables RNase Y to suppress the phenotypes of a ΔcshA mutant.(A) In the left panel, over-night cultures of single mutants ΔcshA, ΔY and YΔ2–24 and double mutants ΔYΔcshA and YΔ2–24ΔcshA were diluted, spotted on agar-plates, and incubated at the indicated temperatures and times. In the right panel, over-night cultures were spotted on horse-blood-agar. (B) Transformants of strain ΔYΔcshA with plasmids expressing variants of RNase Y were selected at 42°C, then restreaked and grown over night at 42°C. Finally the cultures were diluted and spotted at the indicated temperatures. ΔYΔcshA with pYΔ2–24 grows significantly better than the other strains at 24°C. (C) Cartoon showing the four versions of RNase Y expressed from the plasmids; wild-type RNase Y (pY), anchorless RNase Y (pYΔ2–24), RNase Y active site mutant (pY367AA), and anchorless RNase Y active site mutant (pYΔ2–24,367AA). (D) The YΔ2–24ΔcshA strain was transformed with the plasmids expressing the wild-type RNase Y, Y367AA or YΔ2–24,367AA. Overnight cultures were diluted, spotted on agar-plates and incubated at the indicated temperatures for the indicated period of time. Both pY and pY367AA inhibit growth at 24°C. (E) Cartoon showing how the wild-type RNase Y and Y367AA can anchor the YΔ2–24 protein back to the membrane, via dimer-formation.
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pgen.1005577.g005: Removal of the membrane anchor enables RNase Y to suppress the phenotypes of a ΔcshA mutant.(A) In the left panel, over-night cultures of single mutants ΔcshA, ΔY and YΔ2–24 and double mutants ΔYΔcshA and YΔ2–24ΔcshA were diluted, spotted on agar-plates, and incubated at the indicated temperatures and times. In the right panel, over-night cultures were spotted on horse-blood-agar. (B) Transformants of strain ΔYΔcshA with plasmids expressing variants of RNase Y were selected at 42°C, then restreaked and grown over night at 42°C. Finally the cultures were diluted and spotted at the indicated temperatures. ΔYΔcshA with pYΔ2–24 grows significantly better than the other strains at 24°C. (C) Cartoon showing the four versions of RNase Y expressed from the plasmids; wild-type RNase Y (pY), anchorless RNase Y (pYΔ2–24), RNase Y active site mutant (pY367AA), and anchorless RNase Y active site mutant (pYΔ2–24,367AA). (D) The YΔ2–24ΔcshA strain was transformed with the plasmids expressing the wild-type RNase Y, Y367AA or YΔ2–24,367AA. Overnight cultures were diluted, spotted on agar-plates and incubated at the indicated temperatures for the indicated period of time. Both pY and pY367AA inhibit growth at 24°C. (E) Cartoon showing how the wild-type RNase Y and Y367AA can anchor the YΔ2–24 protein back to the membrane, via dimer-formation.

Mentions: The YΔ2–24 mutant was examined by EMOTE, and it became immediately apparent that the membrane anchor is not a requirement for RNase Y enzymatic activity. Every one of the 99 identified RNase Y cleavage sites exhibited a signal in the YΔ2–24 EMOTE data-set (S3 Table), providing evidence that the YΔ2–24 protein is capable of cleaving the same sites as wild-type RNase Y, and in contrast to the ΔY mutant, the hemolysis of the YΔ2–24 mutant is not enhanced (Fig 5A), and the agr mRNA levels are actually decreased (S3D 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)

Removal of the membrane anchor enables RNase Y to suppress the phenotypes of a ΔcshA mutant.(A) In the left panel, over-night cultures of single mutants ΔcshA, ΔY and YΔ2–24 and double mutants ΔYΔcshA and YΔ2–24ΔcshA were diluted, spotted on agar-plates, and incubated at the indicated temperatures and times. In the right panel, over-night cultures were spotted on horse-blood-agar. (B) Transformants of strain ΔYΔcshA with plasmids expressing variants of RNase Y were selected at 42°C, then restreaked and grown over night at 42°C. Finally the cultures were diluted and spotted at the indicated temperatures. ΔYΔcshA with pYΔ2–24 grows significantly better than the other strains at 24°C. (C) Cartoon showing the four versions of RNase Y expressed from the plasmids; wild-type RNase Y (pY), anchorless RNase Y (pYΔ2–24), RNase Y active site mutant (pY367AA), and anchorless RNase Y active site mutant (pYΔ2–24,367AA). (D) The YΔ2–24ΔcshA strain was transformed with the plasmids expressing the wild-type RNase Y, Y367AA or YΔ2–24,367AA. Overnight cultures were diluted, spotted on agar-plates and incubated at the indicated temperatures for the indicated period of time. Both pY and pY367AA inhibit growth at 24°C. (E) Cartoon showing how the wild-type RNase Y and Y367AA can anchor the YΔ2–24 protein back to the membrane, via dimer-formation.
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pgen.1005577.g005: Removal of the membrane anchor enables RNase Y to suppress the phenotypes of a ΔcshA mutant.(A) In the left panel, over-night cultures of single mutants ΔcshA, ΔY and YΔ2–24 and double mutants ΔYΔcshA and YΔ2–24ΔcshA were diluted, spotted on agar-plates, and incubated at the indicated temperatures and times. In the right panel, over-night cultures were spotted on horse-blood-agar. (B) Transformants of strain ΔYΔcshA with plasmids expressing variants of RNase Y were selected at 42°C, then restreaked and grown over night at 42°C. Finally the cultures were diluted and spotted at the indicated temperatures. ΔYΔcshA with pYΔ2–24 grows significantly better than the other strains at 24°C. (C) Cartoon showing the four versions of RNase Y expressed from the plasmids; wild-type RNase Y (pY), anchorless RNase Y (pYΔ2–24), RNase Y active site mutant (pY367AA), and anchorless RNase Y active site mutant (pYΔ2–24,367AA). (D) The YΔ2–24ΔcshA strain was transformed with the plasmids expressing the wild-type RNase Y, Y367AA or YΔ2–24,367AA. Overnight cultures were diluted, spotted on agar-plates and incubated at the indicated temperatures for the indicated period of time. Both pY and pY367AA inhibit growth at 24°C. (E) Cartoon showing how the wild-type RNase Y and Y367AA can anchor the YΔ2–24 protein back to the membrane, via dimer-formation.
Mentions: The YΔ2–24 mutant was examined by EMOTE, and it became immediately apparent that the membrane anchor is not a requirement for RNase Y enzymatic activity. Every one of the 99 identified RNase Y cleavage sites exhibited a signal in the YΔ2–24 EMOTE data-set (S3 Table), providing evidence that the YΔ2–24 protein is capable of cleaving the same sites as wild-type RNase Y, and in contrast to the ΔY mutant, the hemolysis of the YΔ2–24 mutant is not enhanced (Fig 5A), and the agr mRNA levels are actually decreased (S3D 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