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The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5'-UTR Variant.

Quereda JJ, Ortega AD, Pucciarelli MG, García-Del Portillo F - PLoS Genet. (2014)

Bottom Line: The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell.Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR.These findings emphasize how 5'-UTR length affects regulation by defined sRNA.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

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Model of the mechanism by which the L. monocytogenes sRNA Rli27 could positively regulate production of the cell wall protein Lmo0514 inside eukaryotic cells.Rli27 production is markedly stimulated in intracellular L. monocytogenes. A similar effect is observed for the lmo0514 long transcript isoform expressed from promoter P2 (blue). Rli27 interaction with the 5′-UTR region located between promoters P1 and P2 could render the Shine-Dalgarno (SD) site (yellow oval) accessible and facilitate Lmo0514 protein translation. The Rli27-binding site is present only in the transcript with the long 5′-UTR (dark blue oval). The model also considers a hypothetical transient state between the occluded and open states of the long 5′-UTR (dashed rectangle). This transient state could be generated by the intervention of other intracellular factors that might help to open or to stabilize the long 5′-UTR for productive ribosome binding and/or Rli27interaction. These putative factors could also promote Lmo0514 translation in the absence of Rli27, as this protein was detected, although at lower levels, in mutant intracellular bacteria that lack this sRNA.
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pgen-1004765-g007: Model of the mechanism by which the L. monocytogenes sRNA Rli27 could positively regulate production of the cell wall protein Lmo0514 inside eukaryotic cells.Rli27 production is markedly stimulated in intracellular L. monocytogenes. A similar effect is observed for the lmo0514 long transcript isoform expressed from promoter P2 (blue). Rli27 interaction with the 5′-UTR region located between promoters P1 and P2 could render the Shine-Dalgarno (SD) site (yellow oval) accessible and facilitate Lmo0514 protein translation. The Rli27-binding site is present only in the transcript with the long 5′-UTR (dark blue oval). The model also considers a hypothetical transient state between the occluded and open states of the long 5′-UTR (dashed rectangle). This transient state could be generated by the intervention of other intracellular factors that might help to open or to stabilize the long 5′-UTR for productive ribosome binding and/or Rli27interaction. These putative factors could also promote Lmo0514 translation in the absence of Rli27, as this protein was detected, although at lower levels, in mutant intracellular bacteria that lack this sRNA.

Mentions: These in vivo experiments based on complementation assays with Rli27 variants supported a mechanism that involves Rli27 binding to the 5′-UTR of the long lmo0514 transcript variant that is upregulated by L. monocytogenes inside eukaryotic cells. Such an interaction could promote translation, which would lead to increased Lmo0514 protein levels (Fig. 7).


The Listeria Small RNA Rli27 Regulates a Cell Wall Protein inside Eukaryotic Cells by Targeting a Long 5'-UTR Variant.

Quereda JJ, Ortega AD, Pucciarelli MG, García-Del Portillo F - PLoS Genet. (2014)

Model of the mechanism by which the L. monocytogenes sRNA Rli27 could positively regulate production of the cell wall protein Lmo0514 inside eukaryotic cells.Rli27 production is markedly stimulated in intracellular L. monocytogenes. A similar effect is observed for the lmo0514 long transcript isoform expressed from promoter P2 (blue). Rli27 interaction with the 5′-UTR region located between promoters P1 and P2 could render the Shine-Dalgarno (SD) site (yellow oval) accessible and facilitate Lmo0514 protein translation. The Rli27-binding site is present only in the transcript with the long 5′-UTR (dark blue oval). The model also considers a hypothetical transient state between the occluded and open states of the long 5′-UTR (dashed rectangle). This transient state could be generated by the intervention of other intracellular factors that might help to open or to stabilize the long 5′-UTR for productive ribosome binding and/or Rli27interaction. These putative factors could also promote Lmo0514 translation in the absence of Rli27, as this protein was detected, although at lower levels, in mutant intracellular bacteria that lack this sRNA.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4214639&req=5

pgen-1004765-g007: Model of the mechanism by which the L. monocytogenes sRNA Rli27 could positively regulate production of the cell wall protein Lmo0514 inside eukaryotic cells.Rli27 production is markedly stimulated in intracellular L. monocytogenes. A similar effect is observed for the lmo0514 long transcript isoform expressed from promoter P2 (blue). Rli27 interaction with the 5′-UTR region located between promoters P1 and P2 could render the Shine-Dalgarno (SD) site (yellow oval) accessible and facilitate Lmo0514 protein translation. The Rli27-binding site is present only in the transcript with the long 5′-UTR (dark blue oval). The model also considers a hypothetical transient state between the occluded and open states of the long 5′-UTR (dashed rectangle). This transient state could be generated by the intervention of other intracellular factors that might help to open or to stabilize the long 5′-UTR for productive ribosome binding and/or Rli27interaction. These putative factors could also promote Lmo0514 translation in the absence of Rli27, as this protein was detected, although at lower levels, in mutant intracellular bacteria that lack this sRNA.
Mentions: These in vivo experiments based on complementation assays with Rli27 variants supported a mechanism that involves Rli27 binding to the 5′-UTR of the long lmo0514 transcript variant that is upregulated by L. monocytogenes inside eukaryotic cells. Such an interaction could promote translation, which would lead to increased Lmo0514 protein levels (Fig. 7).

Bottom Line: The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell.Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR.These findings emphasize how 5'-UTR length affects regulation by defined sRNA.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Listeria monocytogenes is a bacterial pathogen whose genome encodes many cell wall proteins that bind covalently to peptidoglycan. Some members of this protein family have a key role in virulence, and recent studies show that some of these, such as Lmo0514, are upregulated in bacteria that colonize eukaryotic cells. The regulatory mechanisms that lead to these changes in cell wall proteins remain poorly characterized. Here we studied the regulation responsible for increased Lmo0514 protein levels in intracellular bacteria. The amount of this protein increased markedly in intracellular bacteria (>200-fold), which greatly exceeded the increase in lmo0514 transcript levels (∼6-fold). Rapid amplification of 5'-cDNA ends (RACE) assays identified two lmo0514 transcripts with 5'-untranslated regions (5'-UTR) of 28 and 234 nucleotides. The transcript containing the long 5'-UTR is upregulated by intracellular bacteria. The 234-nucleotide 5'-UTR is also the target of a small RNA (sRNA) denoted Rli27, which we identified by bioinformatics analysis as having extensive base pairing potential with the long 5'-UTR. The interaction is predicted to increase accessibility of the Shine-Dalgarno sequence occluded in the long 5'-UTR and thus to promote Lmo0514 protein production inside the eukaryotic cell. Real-time quantitative PCR showed that Rli27 is upregulated in intracellular bacteria. In vivo experiments indicated a decrease in Lmo0514 protein levels in intracellular bacteria that lacked Rli27. Wild-type Lmo0514 levels were restored by expressing the wild-type Rli27 molecule but not a mutated version unable to interact with the lmo0514 long 5'-UTR. These findings emphasize how 5'-UTR length affects regulation by defined sRNA. In addition, they demonstrate how alterations in the relative abundance of two transcripts with distinct 5'-UTR confine the action of an sRNA for a specific target to bacteria that occupy the intracellular eukaryotic niche.

Show MeSH
Related in: MedlinePlus