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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

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).
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f0001: 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).

Mentions: The Lacto-rpoB motif was first identified by bioinformatics analysis in Lactobacillales, always in the 5' UTR of the rpoB encoding an RNA polymerase subunit, and was suggested to participate in the regulation of rpoB expression.57 In strain SF370, Lacto-rpoB is located between, and in the same orientation as, pbp1b and rpoB and we predicted a putative promoter and a terminator for this element (Fig. 1). We observed several transcript forms of Lacto-rpoB by Northern blot analysis. We visualize 2 long transcripts, and an additional small transcript (<42 nt) present in the wild type and Δrny (RNase Y deletion) strains but absent in the Δrnc (RNase III deletion) mutant (Fig. 1). The small processed transcript (<42 nt) could correspond to the RNase IIIcleavage product of the long primary transcript of the Lacto-rpoB element. We predicted the structure of the Lacto-rpoB motif using RNAfold webserver58,59 and observed that this RNA forms a well-structured RNA with dsRNA regions (Fig. 1). Internal 5' and 3' ends obtained in sRNA sequencing data were positioned on the structure and we observed that these ends could correspond to the 2 nt 3' overhang characteristic of RNase III cleavage (Fig. 1). RNase III cleavage of Lacto-rpoB, based on structure prediction and sRNA sequencing data, would lead to the formation of a 29 nt transcript, corresponding to the <42 nt transcript detected by Northern blot analysis.Figure 1.


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)

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).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829319&req=5

f0001: 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).
Mentions: The Lacto-rpoB motif was first identified by bioinformatics analysis in Lactobacillales, always in the 5' UTR of the rpoB encoding an RNA polymerase subunit, and was suggested to participate in the regulation of rpoB expression.57 In strain SF370, Lacto-rpoB is located between, and in the same orientation as, pbp1b and rpoB and we predicted a putative promoter and a terminator for this element (Fig. 1). We observed several transcript forms of Lacto-rpoB by Northern blot analysis. We visualize 2 long transcripts, and an additional small transcript (<42 nt) present in the wild type and Δrny (RNase Y deletion) strains but absent in the Δrnc (RNase III deletion) mutant (Fig. 1). The small processed transcript (<42 nt) could correspond to the RNase IIIcleavage product of the long primary transcript of the Lacto-rpoB element. We predicted the structure of the Lacto-rpoB motif using RNAfold webserver58,59 and observed that this RNA forms a well-structured RNA with dsRNA regions (Fig. 1). Internal 5' and 3' ends obtained in sRNA sequencing data were positioned on the structure and we observed that these ends could correspond to the 2 nt 3' overhang characteristic of RNase III cleavage (Fig. 1). RNase III cleavage of Lacto-rpoB, based on structure prediction and sRNA sequencing data, would lead to the formation of a 29 nt transcript, corresponding to the <42 nt transcript detected by Northern blot analysis.Figure 1.

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