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RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

Le Rhun A, Beer YY, Reimegård J, Chylinski K, Charpentier E - RNA Biol (2016)

Bottom Line: In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively.Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes.We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

View Article: PubMed Central - PubMed

Affiliation: a The Laboratory for Molecular Infection Sweden (MIMS), Umeå Center for Microbial Research (UCMR), Department of Molecular Biology; Umeå University, S-90187 , Umeå , Sweden.

ABSTRACT
Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

No MeSH data available.


Related in: MedlinePlus

Expression profiles of selected sRNAs. For each sRNA, the locus is depicted with the sRNA in green and the surrounding genes in gray. The prophage regions are indicated in purple. The RNA sequencing expression profiles are captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The putative promoters and terminators are indicated in black. Northern blot analysis (PAGE) were performed in WT (SF370) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. The 5S rRNA is used as a loading control. Folding for the sRNAs was predicted using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrow in (A) represent a putative cleavage site in the RNA. See also Figures S4 and S5 for additional information regarding sRNA sequencing (expression profiles by Northern blot and RNA sequencing analyses, sequence conservation).
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f0002: Expression profiles of selected sRNAs. For each sRNA, the locus is depicted with the sRNA in green and the surrounding genes in gray. The prophage regions are indicated in purple. The RNA sequencing expression profiles are captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The putative promoters and terminators are indicated in black. Northern blot analysis (PAGE) were performed in WT (SF370) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. The 5S rRNA is used as a loading control. Folding for the sRNAs was predicted using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrow in (A) represent a putative cleavage site in the RNA. See also Figures S4 and S5 for additional information regarding sRNA sequencing (expression profiles by Northern blot and RNA sequencing analyses, sequence conservation).

Mentions: For a detailed analysis, we selected 4 distinct sRNAs as examples, Spy_sRNA779816, Spy_sRNA1186876, Spy_sRNA1212757 and Spy_sRNA1786666, that have both a predicted promoter element and a predicted terminator, and represent different lengths and regions (i.e both prophage and non-prophage regions) (Fig. 2, Fig. S5). Their expression was confirmed by Northern blot analysis using total RNA extracted from S. pyogenes SF370 cultured at different growth phases (early logarithmic, mid logarithmic and early stationary phases), and their secondary structures were predicted with RNAfold58,59 (Fig. 2, Fig. S5).Figure 2.


RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes.

Le Rhun A, Beer YY, Reimegård J, Chylinski K, Charpentier E - RNA Biol (2016)

Expression profiles of selected sRNAs. For each sRNA, the locus is depicted with the sRNA in green and the surrounding genes in gray. The prophage regions are indicated in purple. The RNA sequencing expression profiles are captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The putative promoters and terminators are indicated in black. Northern blot analysis (PAGE) were performed in WT (SF370) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. The 5S rRNA is used as a loading control. Folding for the sRNAs was predicted using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrow in (A) represent a putative cleavage site in the RNA. See also Figures S4 and S5 for additional information regarding sRNA sequencing (expression profiles by Northern blot and RNA sequencing analyses, sequence conservation).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829319&req=5

f0002: Expression profiles of selected sRNAs. For each sRNA, the locus is depicted with the sRNA in green and the surrounding genes in gray. The prophage regions are indicated in purple. The RNA sequencing expression profiles are captured using the Integrative Genomics Viewer (IGV) software. The sequence coverage was calculated using BEDTools-Version-2.15.0 and the scale is given in number of reads per million. The putative promoters and terminators are indicated in black. Northern blot analysis (PAGE) were performed in WT (SF370) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. The 5S rRNA is used as a loading control. Folding for the sRNAs was predicted using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrow in (A) represent a putative cleavage site in the RNA. See also Figures S4 and S5 for additional information regarding sRNA sequencing (expression profiles by Northern blot and RNA sequencing analyses, sequence conservation).
Mentions: For a detailed analysis, we selected 4 distinct sRNAs as examples, Spy_sRNA779816, Spy_sRNA1186876, Spy_sRNA1212757 and Spy_sRNA1786666, that have both a predicted promoter element and a predicted terminator, and represent different lengths and regions (i.e both prophage and non-prophage regions) (Fig. 2, Fig. S5). Their expression was confirmed by Northern blot analysis using total RNA extracted from S. pyogenes SF370 cultured at different growth phases (early logarithmic, mid logarithmic and early stationary phases), and their secondary structures were predicted with RNAfold58,59 (Fig. 2, Fig. S5).Figure 2.

Bottom Line: In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively.Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes.We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

View Article: PubMed Central - PubMed

Affiliation: a The Laboratory for Molecular Infection Sweden (MIMS), Umeå Center for Microbial Research (UCMR), Department of Molecular Biology; Umeå University, S-90187 , Umeå , Sweden.

ABSTRACT
Streptococcus pyogenes is a human pathogen responsible for a wide spectrum of diseases ranging from mild to life-threatening infections. During the infectious process, the temporal and spatial expression of pathogenicity factors is tightly controlled by a complex network of protein and RNA regulators acting in response to various environmental signals. Here, we focus on the class of small RNA regulators (sRNAs) and present the first complete analysis of sRNA sequencing data in S. pyogenes. In the SF370 clinical isolate (M1 serotype), we identified 197 and 428 putative regulatory RNAs by visual inspection and bioinformatics screening of the sequencing data, respectively. Only 35 from the 197 candidates identified by visual screening were assigned a predicted function (T-boxes, ribosomal protein leaders, characterized riboswitches or sRNAs), indicating how little is known about sRNA regulation in S. pyogenes. By comparing our list of predicted sRNAs with previous S. pyogenes sRNA screens using bioinformatics or microarrays, 92 novel sRNAs were revealed, including antisense RNAs that are for the first time shown to be expressed in this pathogen. We experimentally validated the expression of 30 novel sRNAs and antisense RNAs. We show that the expression profile of 9 sRNAs including 2 predicted regulatory elements is affected by the endoribonucleases RNase III and/or RNase Y, highlighting the critical role of these enzymes in sRNA regulation.

No MeSH data available.


Related in: MedlinePlus