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Identification and characterization of small RNAs in Yersinia pestis.

Beauregard A, Smith EA, Petrone BL, Singh N, Karch C, McDonough KA, Wade JT - RNA Biol (2013)

Bottom Line: The majority of these sRNAs are not conserved outside the Yersiniae.Expression of the sRNAs was confirmed by Northern analysis and we developed deep sequencing approaches to map 5' and 3' ends of many sRNAs simultaneously.Expression of the majority of the sRNAs we identified is dependent upon Hfq.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center; New York State Department of Health; Albany, NY USA.

ABSTRACT
Yersinia pestis, the etiologic agent of plague, is closely related to Yersinia pseudotuberculosis evolutionarily but has a very different mode of infection. The RNA-binding regulatory protein, Hfq, mediates regulation by small RNAs (sRNAs) and is required for virulence of both Y. pestis and Y. pseudotuberculosis. Moreover, Hfq is required for growth of Y. pestis, but not Y. pseudotuberculosis, at 37°C. Together, these observations suggest that sRNAs play important roles in the virulence and survival of Y. pestis, and that regulation by sRNAs may account for some of the differences between Y. pestis and Y. pseudotuberculosis. We have used a deep sequencing approach to identify 31 sRNAs in Y. pestis. The majority of these sRNAs are not conserved outside the Yersiniae. Expression of the sRNAs was confirmed by Northern analysis and we developed deep sequencing approaches to map 5' and 3' ends of many sRNAs simultaneously. Expression of the majority of the sRNAs we identified is dependent upon Hfq. We also observed temperature-dependent effects on the expression of many sRNAs, and differences in expression patterns between Y. pestis and Y. pseudotuberculosis. Thus, our data suggest that regulation by sRNAs plays an important role in the lifestyle switch from flea to mammalian host, and that regulation by sRNAs may contribute to the phenotypic differences between Y. pestis and Y. pseudotuberculosis.

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Figure 1. (A) Verification of Ysr expression by northern blot analysis. All northern blots are shown in Figure S1 and duplicate northern blots for most sRNAs are shown in Figure S4. A northern blot for hfq mRNA from a corresponding set of RNA samples is also shown (note that this blot is reproduced as part of Fig. S2). Corresponding 5S rRNA northern blots for four of the same membranes are shown in Figure S6. (B) Clusters of sRNA based on k-means clustering of sRNA expression data. Rows correspond to individual RNAs, while columns correspond to conditions for which expression was measured by northern blot. Shading indicates the relative expression of sRNAs for each strain/condition. Expression numbers are indicated as a percentage of the level for condition in which the RNA level is highest. (C) Northern analysis of Ysr170, Ysr172 and Ysr179/CsrB in additional strains of Y. pestis and Y. pseudotuberculosis. Duplicate northern blots are shown in Figure S5. Note that Y. pseudotuberculosis PTB52c is the same strain as used in (A).
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Figure 1: Figure 1. (A) Verification of Ysr expression by northern blot analysis. All northern blots are shown in Figure S1 and duplicate northern blots for most sRNAs are shown in Figure S4. A northern blot for hfq mRNA from a corresponding set of RNA samples is also shown (note that this blot is reproduced as part of Fig. S2). Corresponding 5S rRNA northern blots for four of the same membranes are shown in Figure S6. (B) Clusters of sRNA based on k-means clustering of sRNA expression data. Rows correspond to individual RNAs, while columns correspond to conditions for which expression was measured by northern blot. Shading indicates the relative expression of sRNAs for each strain/condition. Expression numbers are indicated as a percentage of the level for condition in which the RNA level is highest. (C) Northern analysis of Ysr170, Ysr172 and Ysr179/CsrB in additional strains of Y. pestis and Y. pseudotuberculosis. Duplicate northern blots are shown in Figure S5. Note that Y. pseudotuberculosis PTB52c is the same strain as used in (A).

Mentions: To confirm the presence of the putative sRNAs, and to determine their expression profiles in Y. pestis and Y. pseudotuberculosis, we isolated RNA from both species at 28°C and 37°C in hfq+ cells, isogenic Δhfq mutants and Δhfq strains complemented with a multi-copy plasmid that encodes hfq.12 We then performed Northern analysis with radiolabeled oligonucleotides designed to probe each sRNA. The deep sequencing data did not provide strand information so sRNAs were first probed on the plus strand, and any that could not be detected were then probed on the minus strand. This approach confirmed that 32 of the 50 putative sRNAs are expressed at a detectable level and have a size consistent with that of an sRNA (< 500 nt). All Northern-confirmed sRNAs are listed in Table 1 and shown in Figure S1. Representative examples of the northern blots are shown in Figure 1A. Confirmed sRNAs were assigned “Ysr” (Yersinia sRNA) names, in accordance with previously identified sRNAs in Y. pseudotuberculosis.23 One putative sRNA is in fact a protein-coding mRNA for the gene rmf (see below). Of the 31 confirmed sRNAs, 14 have not been described previously, and of the remaining 17, only five have been detected by a method other than deep sequencing.23


Identification and characterization of small RNAs in Yersinia pestis.

Beauregard A, Smith EA, Petrone BL, Singh N, Karch C, McDonough KA, Wade JT - RNA Biol (2013)

Figure 1. (A) Verification of Ysr expression by northern blot analysis. All northern blots are shown in Figure S1 and duplicate northern blots for most sRNAs are shown in Figure S4. A northern blot for hfq mRNA from a corresponding set of RNA samples is also shown (note that this blot is reproduced as part of Fig. S2). Corresponding 5S rRNA northern blots for four of the same membranes are shown in Figure S6. (B) Clusters of sRNA based on k-means clustering of sRNA expression data. Rows correspond to individual RNAs, while columns correspond to conditions for which expression was measured by northern blot. Shading indicates the relative expression of sRNAs for each strain/condition. Expression numbers are indicated as a percentage of the level for condition in which the RNA level is highest. (C) Northern analysis of Ysr170, Ysr172 and Ysr179/CsrB in additional strains of Y. pestis and Y. pseudotuberculosis. Duplicate northern blots are shown in Figure S5. Note that Y. pseudotuberculosis PTB52c is the same strain as used in (A).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672283&req=5

Figure 1: Figure 1. (A) Verification of Ysr expression by northern blot analysis. All northern blots are shown in Figure S1 and duplicate northern blots for most sRNAs are shown in Figure S4. A northern blot for hfq mRNA from a corresponding set of RNA samples is also shown (note that this blot is reproduced as part of Fig. S2). Corresponding 5S rRNA northern blots for four of the same membranes are shown in Figure S6. (B) Clusters of sRNA based on k-means clustering of sRNA expression data. Rows correspond to individual RNAs, while columns correspond to conditions for which expression was measured by northern blot. Shading indicates the relative expression of sRNAs for each strain/condition. Expression numbers are indicated as a percentage of the level for condition in which the RNA level is highest. (C) Northern analysis of Ysr170, Ysr172 and Ysr179/CsrB in additional strains of Y. pestis and Y. pseudotuberculosis. Duplicate northern blots are shown in Figure S5. Note that Y. pseudotuberculosis PTB52c is the same strain as used in (A).
Mentions: To confirm the presence of the putative sRNAs, and to determine their expression profiles in Y. pestis and Y. pseudotuberculosis, we isolated RNA from both species at 28°C and 37°C in hfq+ cells, isogenic Δhfq mutants and Δhfq strains complemented with a multi-copy plasmid that encodes hfq.12 We then performed Northern analysis with radiolabeled oligonucleotides designed to probe each sRNA. The deep sequencing data did not provide strand information so sRNAs were first probed on the plus strand, and any that could not be detected were then probed on the minus strand. This approach confirmed that 32 of the 50 putative sRNAs are expressed at a detectable level and have a size consistent with that of an sRNA (< 500 nt). All Northern-confirmed sRNAs are listed in Table 1 and shown in Figure S1. Representative examples of the northern blots are shown in Figure 1A. Confirmed sRNAs were assigned “Ysr” (Yersinia sRNA) names, in accordance with previously identified sRNAs in Y. pseudotuberculosis.23 One putative sRNA is in fact a protein-coding mRNA for the gene rmf (see below). Of the 31 confirmed sRNAs, 14 have not been described previously, and of the remaining 17, only five have been detected by a method other than deep sequencing.23

Bottom Line: The majority of these sRNAs are not conserved outside the Yersiniae.Expression of the sRNAs was confirmed by Northern analysis and we developed deep sequencing approaches to map 5' and 3' ends of many sRNAs simultaneously.Expression of the majority of the sRNAs we identified is dependent upon Hfq.

View Article: PubMed Central - PubMed

Affiliation: Wadsworth Center; New York State Department of Health; Albany, NY USA.

ABSTRACT
Yersinia pestis, the etiologic agent of plague, is closely related to Yersinia pseudotuberculosis evolutionarily but has a very different mode of infection. The RNA-binding regulatory protein, Hfq, mediates regulation by small RNAs (sRNAs) and is required for virulence of both Y. pestis and Y. pseudotuberculosis. Moreover, Hfq is required for growth of Y. pestis, but not Y. pseudotuberculosis, at 37°C. Together, these observations suggest that sRNAs play important roles in the virulence and survival of Y. pestis, and that regulation by sRNAs may account for some of the differences between Y. pestis and Y. pseudotuberculosis. We have used a deep sequencing approach to identify 31 sRNAs in Y. pestis. The majority of these sRNAs are not conserved outside the Yersiniae. Expression of the sRNAs was confirmed by Northern analysis and we developed deep sequencing approaches to map 5' and 3' ends of many sRNAs simultaneously. Expression of the majority of the sRNAs we identified is dependent upon Hfq. We also observed temperature-dependent effects on the expression of many sRNAs, and differences in expression patterns between Y. pestis and Y. pseudotuberculosis. Thus, our data suggest that regulation by sRNAs plays an important role in the lifestyle switch from flea to mammalian host, and that regulation by sRNAs may contribute to the phenotypic differences between Y. pestis and Y. pseudotuberculosis.

Show MeSH
Related in: MedlinePlus