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. 2016;13(2):177-95.
doi: 10.1080/15476286.2015.1110674.

RNA Sequencing Uncovers Antisense RNAs and Novel Small RNAs in Streptococcus Pyogenes

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Free PMC article

RNA Sequencing Uncovers Antisense RNAs and Novel Small RNAs in Streptococcus Pyogenes

Anaïs Le Rhun et al. RNA Biol. .
Free PMC article

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.

Keywords: Antisense RNAs; RNA sequencing; Streptococcus pyogenes; T-boxes; gene expression regulation; leader RNAs; riboswitches; small RNAs.

Figures

Figure 1.
Figure 1.
For figure legend see page .Figure 1. (see previous page) Lacto-rpoB and 23S methyl RNA elements are regulated by RNase III in S. pyogenes. A. Northern blot analysis (polyacrylamide gel electrophoresis) of Lacto-rpoB and 23S-methyl RNA expression in WT (SF370), ΔRNase III (Δrnc) and ΔRNase Y (Δrny) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. 5S rRNA is used as loading control. B. Expression profiles of Lacto-rpoB and the 23S-methyl motif with surrounding genes 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 distribution of reads starting (5') and ending (3') at each nucleotide position is represented in blue and orange, respectively. The position of the oligonucleotide probes (OLEC) used in Northern blot analysis is indicated. C. Prediction of RNA secondary structure using RNAfold (rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). The arrows represent putative cleavages by RNase III (nucleotides determined by analyzing the 5' and 3' ends of the sRNAs in sRNA sequencing data).
Figure 2.
Figure 2.
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).
Figure 3.
Figure 3.
Expression profiles of sRNAs regulated by RNases. Northern blot analysis of selected sRNAs showing a variation in expression or processing between WT (SF370), ΔRNase III (Δrnc) and ΔRNase Y (Δrny) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. 5S rRNA is used as a loading control. 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. For a detailed set of Northern blots and sRNA sequencing expression patterns, refer to Figure S4.
Figure 4.
Figure 4.
Expression profiles of selected asRNAs. For each asRNA, the locus is depicted with the asRNA in green and the antisense 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 (polyacrylamide gel electrophoresis) was 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 below the sequence coverage. 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). See also Figures S6, S7 and S8 for additional information regarding sRNAs.
Figure 5.
Figure 5.
Expression profiles of asRNAs regulated by RNases. Northern blot analysis of selected asRNAs showing a variation in expression or processing between WT (SF370), ΔRNase III (Δrnc) and ΔRNase Y (Δrny) strains grown to early logarithmic (EL), mid logarithmic (ML) and early stationary (ES) phases. 5S rRNA is used as a loading control. For a detailed set of Northern blots and sRNA sequencing expression patterns, refer to Figure S6.

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