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Novel regulatory small RNAs in Streptococcus pyogenes.

Tesorero RA, Yu N, Wright JO, Svencionis JP, Cheng Q, Kim JH, Cho KH - PLoS ONE (2013)

Bottom Line: Through this process, we discovered 7 putative novel trans-acting sRNAs.Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals.Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Southern Illinois University, Carbondale, Illinois, United States of America.

ABSTRACT
Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes.

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Related in: MedlinePlus

Sequence analysis of candidate sRNA transcriptional start and stop sites, promoter regions and terminators.The transcriptional start and stop sites of candidate sRNAs were determined by circular RACE. The sRNA sequences based on the transcriptional start and stop sites are in black. The putative −10 and −35 promoter sequences are colored green, and putative Rho-independent terminators, which are identified by the algorithm ARNold (http://rna.igmors.u-psud.fr/toolbox/arnold/index.php), are underlined. Neighboring sequences of the sRNA sequences are colored in blue. The deleted part in the SSRC21 deletion mutant, ΔSSRC21cat, is italicized. A putative CovR-binding site upstream of SSRC34 is colored in red. The nucleotide coordinates based on the genome sequence of S. pyogenes MGAS315 and sizes of the sRNAs are shown in parenthesis.
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pone-0064021-g003: Sequence analysis of candidate sRNA transcriptional start and stop sites, promoter regions and terminators.The transcriptional start and stop sites of candidate sRNAs were determined by circular RACE. The sRNA sequences based on the transcriptional start and stop sites are in black. The putative −10 and −35 promoter sequences are colored green, and putative Rho-independent terminators, which are identified by the algorithm ARNold (http://rna.igmors.u-psud.fr/toolbox/arnold/index.php), are underlined. Neighboring sequences of the sRNA sequences are colored in blue. The deleted part in the SSRC21 deletion mutant, ΔSSRC21cat, is italicized. A putative CovR-binding site upstream of SSRC34 is colored in red. The nucleotide coordinates based on the genome sequence of S. pyogenes MGAS315 and sizes of the sRNAs are shown in parenthesis.

Mentions: We performed circular RACE (Rapid Amplification of cDNA Ends) to determine the 5′ and 3′ ends of selected small RNA candidates. Based on the calculated sizes, Northern blot probe-binding sites, and/or putative transcriptional signatures (promoters and Rho-independent terminators), we designed primers for each SSRC. From this analysis, we could determine the sequences of SSRC8, SSRC10, SSRC21, SSRC29, SSRC34 and SSRC41 (Figure 3). All the determined sequences contained a Rho-independent transcriptional termination signal (hairpin structure ending with or followed by thymidines). By examining the sequence of promoter regions, we could map putative −10 and −35 promoter sequences of SSRC10, SSRC21, SSRC29, and a putative CovR-binding sequence [38] upstream of SSRC34 (Figure 3).


Novel regulatory small RNAs in Streptococcus pyogenes.

Tesorero RA, Yu N, Wright JO, Svencionis JP, Cheng Q, Kim JH, Cho KH - PLoS ONE (2013)

Sequence analysis of candidate sRNA transcriptional start and stop sites, promoter regions and terminators.The transcriptional start and stop sites of candidate sRNAs were determined by circular RACE. The sRNA sequences based on the transcriptional start and stop sites are in black. The putative −10 and −35 promoter sequences are colored green, and putative Rho-independent terminators, which are identified by the algorithm ARNold (http://rna.igmors.u-psud.fr/toolbox/arnold/index.php), are underlined. Neighboring sequences of the sRNA sequences are colored in blue. The deleted part in the SSRC21 deletion mutant, ΔSSRC21cat, is italicized. A putative CovR-binding site upstream of SSRC34 is colored in red. The nucleotide coordinates based on the genome sequence of S. pyogenes MGAS315 and sizes of the sRNAs are shown in parenthesis.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3675131&req=5

pone-0064021-g003: Sequence analysis of candidate sRNA transcriptional start and stop sites, promoter regions and terminators.The transcriptional start and stop sites of candidate sRNAs were determined by circular RACE. The sRNA sequences based on the transcriptional start and stop sites are in black. The putative −10 and −35 promoter sequences are colored green, and putative Rho-independent terminators, which are identified by the algorithm ARNold (http://rna.igmors.u-psud.fr/toolbox/arnold/index.php), are underlined. Neighboring sequences of the sRNA sequences are colored in blue. The deleted part in the SSRC21 deletion mutant, ΔSSRC21cat, is italicized. A putative CovR-binding site upstream of SSRC34 is colored in red. The nucleotide coordinates based on the genome sequence of S. pyogenes MGAS315 and sizes of the sRNAs are shown in parenthesis.
Mentions: We performed circular RACE (Rapid Amplification of cDNA Ends) to determine the 5′ and 3′ ends of selected small RNA candidates. Based on the calculated sizes, Northern blot probe-binding sites, and/or putative transcriptional signatures (promoters and Rho-independent terminators), we designed primers for each SSRC. From this analysis, we could determine the sequences of SSRC8, SSRC10, SSRC21, SSRC29, SSRC34 and SSRC41 (Figure 3). All the determined sequences contained a Rho-independent transcriptional termination signal (hairpin structure ending with or followed by thymidines). By examining the sequence of promoter regions, we could map putative −10 and −35 promoter sequences of SSRC10, SSRC21, SSRC29, and a putative CovR-binding sequence [38] upstream of SSRC34 (Figure 3).

Bottom Line: Through this process, we discovered 7 putative novel trans-acting sRNAs.Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals.Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Southern Illinois University, Carbondale, Illinois, United States of America.

ABSTRACT
Streptococcus pyogenes (Group A Streptococcus or GAS) is a Gram-positive bacterial pathogen that has shown complex modes of regulation of its virulence factors to cause diverse diseases. Bacterial small RNAs are regarded as novel widespread regulators of gene expression in response to environmental signals. Recent studies have revealed that several small RNAs (sRNAs) have an important role in S. pyogenes physiology and pathogenesis by regulating gene expression at the translational level. To search for new sRNAs in S. pyogenes, we performed a genomewide analysis through computational prediction followed by experimental verification. To overcome the limitation of low accuracy in computational prediction, we employed a combination of three different computational algorithms (sRNAPredict, eQRNA and RNAz). A total of 45 candidates were chosen based on the computational analysis, and their transcription was analyzed by reverse-transcriptase PCR and Northern blot. Through this process, we discovered 7 putative novel trans-acting sRNAs. Their abundance varied between different growth phases, suggesting that their expression is influenced by environmental or internal signals. Further, to screen target mRNAs of an sRNA, we employed differential RNA sequencing analysis. This study provides a significant resource for future study of small RNAs and their roles in physiology and pathogenesis of S. pyogenes.

Show MeSH
Related in: MedlinePlus