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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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Rli27 interacts in vitro with the lmo0514 long 5′-UTR.(A) Scheme of the major interaction region between Rli27 and the lmo0514 5′-UTR predicted with the targetRNA program (http://cs.wellesley.edu/~btjaden/TargetRNA2/). The complete set of putative interaction sites is shown in Fig. S3. (B) Effect of Rli27-mut1 and Rli27-mut3 mutations on the predicted Rli27-5′-UTR-lmo0514 interaction. Changes are highlighted in yellow. Compensatory mutations designed in 5′-UTR molecules synthesized in vitro are also shown in Fig. S3. (C) EMSA assays showing formation of a 5′-UTR-lmo0514/Rli27 duplex with slow migration in the gel. This duplex is not formed after co-incubation of the 5′-UTR molecule with the Rli27-mut1 or Rli27-mut3 variants, and is partially restored by compensatory mutations in the lmo0514 5′-UTR. (D) Control EMSA showing no duplex formation after incubation of the lmo0514 5′-UTR with an unrelated sRNA, SbrA.
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pgen-1004765-g005: Rli27 interacts in vitro with the lmo0514 long 5′-UTR.(A) Scheme of the major interaction region between Rli27 and the lmo0514 5′-UTR predicted with the targetRNA program (http://cs.wellesley.edu/~btjaden/TargetRNA2/). The complete set of putative interaction sites is shown in Fig. S3. (B) Effect of Rli27-mut1 and Rli27-mut3 mutations on the predicted Rli27-5′-UTR-lmo0514 interaction. Changes are highlighted in yellow. Compensatory mutations designed in 5′-UTR molecules synthesized in vitro are also shown in Fig. S3. (C) EMSA assays showing formation of a 5′-UTR-lmo0514/Rli27 duplex with slow migration in the gel. This duplex is not formed after co-incubation of the 5′-UTR molecule with the Rli27-mut1 or Rli27-mut3 variants, and is partially restored by compensatory mutations in the lmo0514 5′-UTR. (D) Control EMSA showing no duplex formation after incubation of the lmo0514 5′-UTR with an unrelated sRNA, SbrA.

Mentions: Rli27 interaction with the lmo0514 5′-UTR extends to several regions, although it shows a major predicted pairing region involving Rli27 nucleotides 1 to 21 (Fig. 5A, Fig. S3). We used electrophoretic mobility shift assays (EMSA) to assess the validity of this prediction. We generated in vitro wild-type versions of Rli27 and 5′-UTR-lmo0514, together with variants of both RNA molecules bearing mutations in 3 nt (mut-1) or 14 nt (mut-3) important for pairing (Fig. 5B). Incubation of Rli27 and 5′-UTR-lmo0514 wild-type molecules resulted in a duplex with low electrophoretic mobility (Fig. 5C). Conversely, combination of wild-type 5′-UTR-lmo0514 with mutated Rli27 (either mut-1 or mut-3 variants), reduced duplex formation (Fig. 5C). Duplex formation was partially restored by combining mutations in Rli27 with compensatory mutations in 5′-UTR-lmo0514 (Fig. 5C). Specificity of the Rli27-5′-UTR-lmo0514 interaction was confirmed by lack of duplex formation after incubation of the 5′-UTR-lmo0514 wild-type molecule with SbrA, an unrelated sRNA (Fig. 5D).


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)

Rli27 interacts in vitro with the lmo0514 long 5′-UTR.(A) Scheme of the major interaction region between Rli27 and the lmo0514 5′-UTR predicted with the targetRNA program (http://cs.wellesley.edu/~btjaden/TargetRNA2/). The complete set of putative interaction sites is shown in Fig. S3. (B) Effect of Rli27-mut1 and Rli27-mut3 mutations on the predicted Rli27-5′-UTR-lmo0514 interaction. Changes are highlighted in yellow. Compensatory mutations designed in 5′-UTR molecules synthesized in vitro are also shown in Fig. S3. (C) EMSA assays showing formation of a 5′-UTR-lmo0514/Rli27 duplex with slow migration in the gel. This duplex is not formed after co-incubation of the 5′-UTR molecule with the Rli27-mut1 or Rli27-mut3 variants, and is partially restored by compensatory mutations in the lmo0514 5′-UTR. (D) Control EMSA showing no duplex formation after incubation of the lmo0514 5′-UTR with an unrelated sRNA, SbrA.
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pgen-1004765-g005: Rli27 interacts in vitro with the lmo0514 long 5′-UTR.(A) Scheme of the major interaction region between Rli27 and the lmo0514 5′-UTR predicted with the targetRNA program (http://cs.wellesley.edu/~btjaden/TargetRNA2/). The complete set of putative interaction sites is shown in Fig. S3. (B) Effect of Rli27-mut1 and Rli27-mut3 mutations on the predicted Rli27-5′-UTR-lmo0514 interaction. Changes are highlighted in yellow. Compensatory mutations designed in 5′-UTR molecules synthesized in vitro are also shown in Fig. S3. (C) EMSA assays showing formation of a 5′-UTR-lmo0514/Rli27 duplex with slow migration in the gel. This duplex is not formed after co-incubation of the 5′-UTR molecule with the Rli27-mut1 or Rli27-mut3 variants, and is partially restored by compensatory mutations in the lmo0514 5′-UTR. (D) Control EMSA showing no duplex formation after incubation of the lmo0514 5′-UTR with an unrelated sRNA, SbrA.
Mentions: Rli27 interaction with the lmo0514 5′-UTR extends to several regions, although it shows a major predicted pairing region involving Rli27 nucleotides 1 to 21 (Fig. 5A, Fig. S3). We used electrophoretic mobility shift assays (EMSA) to assess the validity of this prediction. We generated in vitro wild-type versions of Rli27 and 5′-UTR-lmo0514, together with variants of both RNA molecules bearing mutations in 3 nt (mut-1) or 14 nt (mut-3) important for pairing (Fig. 5B). Incubation of Rli27 and 5′-UTR-lmo0514 wild-type molecules resulted in a duplex with low electrophoretic mobility (Fig. 5C). Conversely, combination of wild-type 5′-UTR-lmo0514 with mutated Rli27 (either mut-1 or mut-3 variants), reduced duplex formation (Fig. 5C). Duplex formation was partially restored by combining mutations in Rli27 with compensatory mutations in 5′-UTR-lmo0514 (Fig. 5C). Specificity of the Rli27-5′-UTR-lmo0514 interaction was confirmed by lack of duplex formation after incubation of the 5′-UTR-lmo0514 wild-type molecule with SbrA, an unrelated sRNA (Fig. 5D).

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