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Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation.

Urban JH, Vogel J - PLoS Biol. (2008)

Bottom Line: We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction.Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA.Similar cascades of noncoding RNA regulators may operate in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Infection Biology, RNA Biology Group, Berlin, Germany.

ABSTRACT
Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms.

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Related in: MedlinePlus

GlmZ Directly Acts on the glmS mRNA to Enhance GlmS Synthesis(A) Predicted anti-antisense mechanism by which GlmZ activates the E. coli glmS mRNA. The left panel shows the 5′ UTR of the glmS mRNA in an unbound, “inactive” state in which an intrinsic hairpin sequesters the glmS SD (boxed) and thus inhibits translation. Residues −42 to +3 relative to the AUG start codon, which is underlined, are shown. The right panel shows glmS mRNA in its “activated” form, i.e., upon base-pairing of residues 150–157 and 163–169 of GlmZ RNA with the glmS 5′ UTR, which disrupts the inhibitory hairpin and liberates the glmS SD. The positions of point mutations introduced in glmS (C−27 → G, G−36 → C; glmS* allele) and GlmZ (G+155 → C, C+165 → G; GlmZ* allele), which are expected to maintain base pairing in the glmS*/GlmZ* duplex, are indicated by inverse (white on black) print.(B) Colony fluorescence of strain JVS-8113 (ΔglmY ΔglmZ) carrying either the glmS::gfp or glmS*::gfp fusion plasmids, each in combination with plasmid pJV300 (“contr”), pPL-GlmZ (“GlmZ”), or pPL-GlmZ* (“GlmZ*”). Shown is an image of a LB agar plate obtained in the fluorescence mode.(C) The strains shown in (B) were grown to early stationary phase and subjected to western blot (top three panels) and northern blot (bottom three panels) analysis. The plasmid-encoded GlmS::GFP fusion protein was detected using α-GFP antibodies (top panel); the chromosomally encoded glmS mRNA and GlmS protein, and the plasmid-encoded GlmZ RNA (as indicated) were detected as in Figure 2.
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pbio-0060064-g003: GlmZ Directly Acts on the glmS mRNA to Enhance GlmS Synthesis(A) Predicted anti-antisense mechanism by which GlmZ activates the E. coli glmS mRNA. The left panel shows the 5′ UTR of the glmS mRNA in an unbound, “inactive” state in which an intrinsic hairpin sequesters the glmS SD (boxed) and thus inhibits translation. Residues −42 to +3 relative to the AUG start codon, which is underlined, are shown. The right panel shows glmS mRNA in its “activated” form, i.e., upon base-pairing of residues 150–157 and 163–169 of GlmZ RNA with the glmS 5′ UTR, which disrupts the inhibitory hairpin and liberates the glmS SD. The positions of point mutations introduced in glmS (C−27 → G, G−36 → C; glmS* allele) and GlmZ (G+155 → C, C+165 → G; GlmZ* allele), which are expected to maintain base pairing in the glmS*/GlmZ* duplex, are indicated by inverse (white on black) print.(B) Colony fluorescence of strain JVS-8113 (ΔglmY ΔglmZ) carrying either the glmS::gfp or glmS*::gfp fusion plasmids, each in combination with plasmid pJV300 (“contr”), pPL-GlmZ (“GlmZ”), or pPL-GlmZ* (“GlmZ*”). Shown is an image of a LB agar plate obtained in the fluorescence mode.(C) The strains shown in (B) were grown to early stationary phase and subjected to western blot (top three panels) and northern blot (bottom three panels) analysis. The plasmid-encoded GlmS::GFP fusion protein was detected using α-GFP antibodies (top panel); the chromosomally encoded glmS mRNA and GlmS protein, and the plasmid-encoded GlmZ RNA (as indicated) were detected as in Figure 2.

Mentions: (A) Consensus structures of the E. coli GlmY (184 nt) and GlmZ (207 nt) RNAs (see [3] and Figure S1, respectively, for sequence alignments). Vertical arrows indicate previously mapped 3′ processing sites [6,9]. Grey circles denote nucleotides conserved between the E. coli GlmY and GlmZ RNAs. The GlmZ residues involved in glmS mRNA binding (see Figure 3A below) are shown in red.


Two seemingly homologous noncoding RNAs act hierarchically to activate glmS mRNA translation.

Urban JH, Vogel J - PLoS Biol. (2008)

GlmZ Directly Acts on the glmS mRNA to Enhance GlmS Synthesis(A) Predicted anti-antisense mechanism by which GlmZ activates the E. coli glmS mRNA. The left panel shows the 5′ UTR of the glmS mRNA in an unbound, “inactive” state in which an intrinsic hairpin sequesters the glmS SD (boxed) and thus inhibits translation. Residues −42 to +3 relative to the AUG start codon, which is underlined, are shown. The right panel shows glmS mRNA in its “activated” form, i.e., upon base-pairing of residues 150–157 and 163–169 of GlmZ RNA with the glmS 5′ UTR, which disrupts the inhibitory hairpin and liberates the glmS SD. The positions of point mutations introduced in glmS (C−27 → G, G−36 → C; glmS* allele) and GlmZ (G+155 → C, C+165 → G; GlmZ* allele), which are expected to maintain base pairing in the glmS*/GlmZ* duplex, are indicated by inverse (white on black) print.(B) Colony fluorescence of strain JVS-8113 (ΔglmY ΔglmZ) carrying either the glmS::gfp or glmS*::gfp fusion plasmids, each in combination with plasmid pJV300 (“contr”), pPL-GlmZ (“GlmZ”), or pPL-GlmZ* (“GlmZ*”). Shown is an image of a LB agar plate obtained in the fluorescence mode.(C) The strains shown in (B) were grown to early stationary phase and subjected to western blot (top three panels) and northern blot (bottom three panels) analysis. The plasmid-encoded GlmS::GFP fusion protein was detected using α-GFP antibodies (top panel); the chromosomally encoded glmS mRNA and GlmS protein, and the plasmid-encoded GlmZ RNA (as indicated) were detected as in Figure 2.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2267818&req=5

pbio-0060064-g003: GlmZ Directly Acts on the glmS mRNA to Enhance GlmS Synthesis(A) Predicted anti-antisense mechanism by which GlmZ activates the E. coli glmS mRNA. The left panel shows the 5′ UTR of the glmS mRNA in an unbound, “inactive” state in which an intrinsic hairpin sequesters the glmS SD (boxed) and thus inhibits translation. Residues −42 to +3 relative to the AUG start codon, which is underlined, are shown. The right panel shows glmS mRNA in its “activated” form, i.e., upon base-pairing of residues 150–157 and 163–169 of GlmZ RNA with the glmS 5′ UTR, which disrupts the inhibitory hairpin and liberates the glmS SD. The positions of point mutations introduced in glmS (C−27 → G, G−36 → C; glmS* allele) and GlmZ (G+155 → C, C+165 → G; GlmZ* allele), which are expected to maintain base pairing in the glmS*/GlmZ* duplex, are indicated by inverse (white on black) print.(B) Colony fluorescence of strain JVS-8113 (ΔglmY ΔglmZ) carrying either the glmS::gfp or glmS*::gfp fusion plasmids, each in combination with plasmid pJV300 (“contr”), pPL-GlmZ (“GlmZ”), or pPL-GlmZ* (“GlmZ*”). Shown is an image of a LB agar plate obtained in the fluorescence mode.(C) The strains shown in (B) were grown to early stationary phase and subjected to western blot (top three panels) and northern blot (bottom three panels) analysis. The plasmid-encoded GlmS::GFP fusion protein was detected using α-GFP antibodies (top panel); the chromosomally encoded glmS mRNA and GlmS protein, and the plasmid-encoded GlmZ RNA (as indicated) were detected as in Figure 2.
Mentions: (A) Consensus structures of the E. coli GlmY (184 nt) and GlmZ (207 nt) RNAs (see [3] and Figure S1, respectively, for sequence alignments). Vertical arrows indicate previously mapped 3′ processing sites [6,9]. Grey circles denote nucleotides conserved between the E. coli GlmY and GlmZ RNAs. The GlmZ residues involved in glmS mRNA binding (see Figure 3A below) are shown in red.

Bottom Line: We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction.Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA.Similar cascades of noncoding RNA regulators may operate in other organisms.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute for Infection Biology, RNA Biology Group, Berlin, Germany.

ABSTRACT
Small noncoding RNAs (sRNA) can function as posttranscriptional activators of gene expression to regulate stress responses and metabolism. We here describe the mechanisms by which two sRNAs, GlmY and GlmZ, activate the Escherichia coli glmS mRNA, coding for an essential enzyme in amino-sugar metabolism. The two sRNAs, although being highly similar in sequence and structure, act in a hierarchical manner. GlmZ, together with the RNA chaperone, Hfq, directly activates glmS mRNA translation by an anti-antisense mechanism. In contrast, GlmY acts upstream of GlmZ and positively regulates glmS by antagonizing GlmZ RNA inactivation. We also report the first example, to our knowledge, of mRNA expression being controlled by the poly(A) status of a chromosomally encoded sRNA. We show that in wild-type cells, GlmY RNA is unstable due to 3' end polyadenylation; whereas in an E. coli pcnB mutant defective in RNA polyadenylation, GlmY is stabilized and accumulates, which in turn stabilizes GlmZ and causes GlmS overproduction. Our study reveals hierarchical action of two well-conserved sRNAs in a complex regulatory cascade that controls the glmS mRNA. Similar cascades of noncoding RNA regulators may operate in other organisms.

Show MeSH
Related in: MedlinePlus