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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 2. Representative examples of 5′ and 3′ Deep RACE data. (A) Schematic for the Deep 5′ RACE method. (B) Schematic for the Deep 3′ RACE method. (C) Deep 5′ RACE data (blue) and Deep 3′ RACE data (green) for four selected sRNAs. Flanking annotated genes are indicated by gray boxes.
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Figure 2: Figure 2. Representative examples of 5′ and 3′ Deep RACE data. (A) Schematic for the Deep 5′ RACE method. (B) Schematic for the Deep 3′ RACE method. (C) Deep 5′ RACE data (blue) and Deep 3′ RACE data (green) for four selected sRNAs. Flanking annotated genes are indicated by gray boxes.

Mentions: We developed genome-scale 5′ and 3′ RACE approaches to precisely determine the ends of the sRNAs confirmed by Northern analysis (Fig. 2A and B) because our deep sequencing data do not allow for such precise mapping. These methods combine conventional RACE with deep sequencing using the Ion Torrent platform (any deep sequencing platform would suffice). In addition to allowing for simultaneous analysis of many RNAs, these methods produce multiple sequence reads for each individual sRNA (e.g., 1,945 sequence reads for Ysr23/160). This allows us to identify multiple 5′ ends and to accurately determine the relative abundance of each. We propose that these methods be named “Deep 5′ RACE” and “Deep 3′ RACE.” Using these methods, we successfully mapped the 5′ ends of 18 sRNAs (and rmf mRNA) and the 3′ ends of 28 sRNAs (and rmf mRNA). The major 5′ and 3′ ends for these sRNAs are listed in Table 1 and raw data are provided in Tables S1 and 2. Four representative examples are shown in Figure 2C‒F. For the sRNAs for which we mapped both unique 5′ and 3′ ends, the median length is 84 nt. In most cases we detected unique ends, but some RNAs have multiple 5′ ends, e.g., Ysr149/181 (Fig. 2E; Table 1). In addition, many sRNAs have multiple 3′ ends clustered around a single location, e.g., Ysr148/153/GlmZ (Fig. 2C), Ysr149/181 (Fig. 2E), Ysr17/154/MicA (Fig. 2F; Table 1). Most sRNAs are located entirely within intergenic regions but some overlap the ends of adjacent genes, e.g., Ysr165 (Fig. 2D; Table 1). Our Deep 5′ RACE method is very similar to a previously described method, “Deep-RACE.”24 To the best of our knowledge, no method equivalent to Deep 3′ RACE has been described previously. Given the increasing availability of deep sequencing, we anticipate that these methods will become widespread for the large-scale identification of RNA 5′ and 3′ ends.


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 2. Representative examples of 5′ and 3′ Deep RACE data. (A) Schematic for the Deep 5′ RACE method. (B) Schematic for the Deep 3′ RACE method. (C) Deep 5′ RACE data (blue) and Deep 3′ RACE data (green) for four selected sRNAs. Flanking annotated genes are indicated by gray boxes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Figure 2. Representative examples of 5′ and 3′ Deep RACE data. (A) Schematic for the Deep 5′ RACE method. (B) Schematic for the Deep 3′ RACE method. (C) Deep 5′ RACE data (blue) and Deep 3′ RACE data (green) for four selected sRNAs. Flanking annotated genes are indicated by gray boxes.
Mentions: We developed genome-scale 5′ and 3′ RACE approaches to precisely determine the ends of the sRNAs confirmed by Northern analysis (Fig. 2A and B) because our deep sequencing data do not allow for such precise mapping. These methods combine conventional RACE with deep sequencing using the Ion Torrent platform (any deep sequencing platform would suffice). In addition to allowing for simultaneous analysis of many RNAs, these methods produce multiple sequence reads for each individual sRNA (e.g., 1,945 sequence reads for Ysr23/160). This allows us to identify multiple 5′ ends and to accurately determine the relative abundance of each. We propose that these methods be named “Deep 5′ RACE” and “Deep 3′ RACE.” Using these methods, we successfully mapped the 5′ ends of 18 sRNAs (and rmf mRNA) and the 3′ ends of 28 sRNAs (and rmf mRNA). The major 5′ and 3′ ends for these sRNAs are listed in Table 1 and raw data are provided in Tables S1 and 2. Four representative examples are shown in Figure 2C‒F. For the sRNAs for which we mapped both unique 5′ and 3′ ends, the median length is 84 nt. In most cases we detected unique ends, but some RNAs have multiple 5′ ends, e.g., Ysr149/181 (Fig. 2E; Table 1). In addition, many sRNAs have multiple 3′ ends clustered around a single location, e.g., Ysr148/153/GlmZ (Fig. 2C), Ysr149/181 (Fig. 2E), Ysr17/154/MicA (Fig. 2F; Table 1). Most sRNAs are located entirely within intergenic regions but some overlap the ends of adjacent genes, e.g., Ysr165 (Fig. 2D; Table 1). Our Deep 5′ RACE method is very similar to a previously described method, “Deep-RACE.”24 To the best of our knowledge, no method equivalent to Deep 3′ RACE has been described previously. Given the increasing availability of deep sequencing, we anticipate that these methods will become widespread for the large-scale identification of RNA 5′ and 3′ ends.

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