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Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B, Martin-Verstraete I - PLoS Genet. (2013)

Bottom Line: Expression of 35 sRNAs was confirmed by gene-specific experimental approaches.These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities.Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France. olga.soutourina@pasteur.fr

ABSTRACT
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

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Effect of RCd2 RNA overexpression on target gene control.(A) Sequence of RCd2 RNA within CD0182 and CD0183 IGR. The TSS “+1” for antisense RNA and CD0183 mRNA identified by 5′-end RNA-seq are indicated by broken arrows. The 5′- and 3′-ends of RCd2 RNA identified by 5′/3′RACE are shown in bold and are indicated by stars and in grey boxes, respectively. The −10 and −35 regions are boxed. The transcriptional terminator for RCd2 is indicated by convergent arrows. The CD0182 and CD0183 start codons and the CD0182 stop codon are shown in black. The ribosome binding site (RBS) of CD0183 is inderlined. The numbers indicate positions relative to the CD0183 TSS. (B) Growth of 630/p strain (triangles) and 630/pRCd2 strain (squares) in TY medium at 37°C in the presence (open symbols) or absence (closed symbols) of 250 ng/mL ATc. (C) Effect of RCd2 overexpression on the CD0183 transcript abundance. For Northern blot analysis, RNA samples were extracted from 630Δerm strain during exponential growth phase (E, 4 h of growth), at the onset of stationary phase (S, 10 h of growth) and from 630/p control strain or from 630/pRCd2 strain overexpressing RCd2 grown at late exponential growth phase (LE) in the presence of 250 ng/mL ATc (630/p, 630/pRCd2). Detection with RCd2-specific probe is shown on the left and with CD0183-specific probe on the right. 5S RNA at the bottom serves as loading control. Longer exposure time was required for better detection of RCd2 transcripts in 630Δerm and 630/p strains. The RNA secondary structure prediction (D) was performed by Mfold software. The terminator at the 3′-end and loop region at the 5′-end overlapping 5′-part of CD0183 mRNA are highlighted in orange.
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pgen-1003493-g006: Effect of RCd2 RNA overexpression on target gene control.(A) Sequence of RCd2 RNA within CD0182 and CD0183 IGR. The TSS “+1” for antisense RNA and CD0183 mRNA identified by 5′-end RNA-seq are indicated by broken arrows. The 5′- and 3′-ends of RCd2 RNA identified by 5′/3′RACE are shown in bold and are indicated by stars and in grey boxes, respectively. The −10 and −35 regions are boxed. The transcriptional terminator for RCd2 is indicated by convergent arrows. The CD0182 and CD0183 start codons and the CD0182 stop codon are shown in black. The ribosome binding site (RBS) of CD0183 is inderlined. The numbers indicate positions relative to the CD0183 TSS. (B) Growth of 630/p strain (triangles) and 630/pRCd2 strain (squares) in TY medium at 37°C in the presence (open symbols) or absence (closed symbols) of 250 ng/mL ATc. (C) Effect of RCd2 overexpression on the CD0183 transcript abundance. For Northern blot analysis, RNA samples were extracted from 630Δerm strain during exponential growth phase (E, 4 h of growth), at the onset of stationary phase (S, 10 h of growth) and from 630/p control strain or from 630/pRCd2 strain overexpressing RCd2 grown at late exponential growth phase (LE) in the presence of 250 ng/mL ATc (630/p, 630/pRCd2). Detection with RCd2-specific probe is shown on the left and with CD0183-specific probe on the right. 5S RNA at the bottom serves as loading control. Longer exposure time was required for better detection of RCd2 transcripts in 630Δerm and 630/p strains. The RNA secondary structure prediction (D) was performed by Mfold software. The terminator at the 3′-end and loop region at the 5′-end overlapping 5′-part of CD0183 mRNA are highlighted in orange.

Mentions: Among the 185 C. difficile 630Δerm sRNAs identified by deep sequencing, 35 out of 40 candidates assayed were detected by Northern blotting of 630Δerm strain RNA samples, while 23 were detected in RNA prepared from the R20291 strain samples. Transcript lengths agreed well with the sizes deduced from RNA-seq approach (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8; Figures S2, S3, S4, S5) and were confirmed by independent 5′/3′RACE analysis for 4 selected RNAs (Table S4). 5′RACE experiments unambiguously identified TSSs for eight potential sRNAs and were in complete agreement with the TSSs identified by 5′-end RNA-seq (Table S4).


Genome-wide identification of regulatory RNAs in the human pathogen Clostridium difficile.

Soutourina OA, Monot M, Boudry P, Saujet L, Pichon C, Sismeiro O, Semenova E, Severinov K, Le Bouguenec C, Coppée JY, Dupuy B, Martin-Verstraete I - PLoS Genet. (2013)

Effect of RCd2 RNA overexpression on target gene control.(A) Sequence of RCd2 RNA within CD0182 and CD0183 IGR. The TSS “+1” for antisense RNA and CD0183 mRNA identified by 5′-end RNA-seq are indicated by broken arrows. The 5′- and 3′-ends of RCd2 RNA identified by 5′/3′RACE are shown in bold and are indicated by stars and in grey boxes, respectively. The −10 and −35 regions are boxed. The transcriptional terminator for RCd2 is indicated by convergent arrows. The CD0182 and CD0183 start codons and the CD0182 stop codon are shown in black. The ribosome binding site (RBS) of CD0183 is inderlined. The numbers indicate positions relative to the CD0183 TSS. (B) Growth of 630/p strain (triangles) and 630/pRCd2 strain (squares) in TY medium at 37°C in the presence (open symbols) or absence (closed symbols) of 250 ng/mL ATc. (C) Effect of RCd2 overexpression on the CD0183 transcript abundance. For Northern blot analysis, RNA samples were extracted from 630Δerm strain during exponential growth phase (E, 4 h of growth), at the onset of stationary phase (S, 10 h of growth) and from 630/p control strain or from 630/pRCd2 strain overexpressing RCd2 grown at late exponential growth phase (LE) in the presence of 250 ng/mL ATc (630/p, 630/pRCd2). Detection with RCd2-specific probe is shown on the left and with CD0183-specific probe on the right. 5S RNA at the bottom serves as loading control. Longer exposure time was required for better detection of RCd2 transcripts in 630Δerm and 630/p strains. The RNA secondary structure prediction (D) was performed by Mfold software. The terminator at the 3′-end and loop region at the 5′-end overlapping 5′-part of CD0183 mRNA are highlighted in orange.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3649979&req=5

pgen-1003493-g006: Effect of RCd2 RNA overexpression on target gene control.(A) Sequence of RCd2 RNA within CD0182 and CD0183 IGR. The TSS “+1” for antisense RNA and CD0183 mRNA identified by 5′-end RNA-seq are indicated by broken arrows. The 5′- and 3′-ends of RCd2 RNA identified by 5′/3′RACE are shown in bold and are indicated by stars and in grey boxes, respectively. The −10 and −35 regions are boxed. The transcriptional terminator for RCd2 is indicated by convergent arrows. The CD0182 and CD0183 start codons and the CD0182 stop codon are shown in black. The ribosome binding site (RBS) of CD0183 is inderlined. The numbers indicate positions relative to the CD0183 TSS. (B) Growth of 630/p strain (triangles) and 630/pRCd2 strain (squares) in TY medium at 37°C in the presence (open symbols) or absence (closed symbols) of 250 ng/mL ATc. (C) Effect of RCd2 overexpression on the CD0183 transcript abundance. For Northern blot analysis, RNA samples were extracted from 630Δerm strain during exponential growth phase (E, 4 h of growth), at the onset of stationary phase (S, 10 h of growth) and from 630/p control strain or from 630/pRCd2 strain overexpressing RCd2 grown at late exponential growth phase (LE) in the presence of 250 ng/mL ATc (630/p, 630/pRCd2). Detection with RCd2-specific probe is shown on the left and with CD0183-specific probe on the right. 5S RNA at the bottom serves as loading control. Longer exposure time was required for better detection of RCd2 transcripts in 630Δerm and 630/p strains. The RNA secondary structure prediction (D) was performed by Mfold software. The terminator at the 3′-end and loop region at the 5′-end overlapping 5′-part of CD0183 mRNA are highlighted in orange.
Mentions: Among the 185 C. difficile 630Δerm sRNAs identified by deep sequencing, 35 out of 40 candidates assayed were detected by Northern blotting of 630Δerm strain RNA samples, while 23 were detected in RNA prepared from the R20291 strain samples. Transcript lengths agreed well with the sizes deduced from RNA-seq approach (Figure 2, Figure 3, Figure 4, Figure 5, Figure 6, Figure 7, Figure 8; Figures S2, S3, S4, S5) and were confirmed by independent 5′/3′RACE analysis for 4 selected RNAs (Table S4). 5′RACE experiments unambiguously identified TSSs for eight potential sRNAs and were in complete agreement with the TSSs identified by 5′-end RNA-seq (Table S4).

Bottom Line: Expression of 35 sRNAs was confirmed by gene-specific experimental approaches.These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities.Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire Pathogenèse des Bactéries Anaérobies, Institut Pasteur, Paris, France. olga.soutourina@pasteur.fr

ABSTRACT
Clostridium difficile is an emergent pathogen, and the most common cause of nosocomial diarrhea. In an effort to understand the role of small noncoding RNAs (sRNAs) in C. difficile physiology and pathogenesis, we used an in silico approach to identify 511 sRNA candidates in both intergenic and coding regions. In parallel, RNA-seq and differential 5'-end RNA-seq were used for global identification of C. difficile sRNAs and their transcriptional start sites at three different growth conditions (exponential growth phase, stationary phase, and starvation). This global experimental approach identified 251 putative regulatory sRNAs including 94 potential trans riboregulators located in intergenic regions, 91 cis-antisense RNAs, and 66 riboswitches. Expression of 35 sRNAs was confirmed by gene-specific experimental approaches. Some sRNAs, including an antisense RNA that may be involved in control of C. difficile autolytic activity, showed growth phase-dependent expression profiles. Expression of each of 16 predicted c-di-GMP-responsive riboswitches was observed, and experimental evidence for their regulatory role in coordinated control of motility and biofilm formation was obtained. Finally, we detected abundant sRNAs encoded by multiple C. difficile CRISPR loci. These RNAs may be important for C. difficile survival in bacteriophage-rich gut communities. Altogether, this first experimental genome-wide identification of C. difficile sRNAs provides a firm basis for future RNome characterization and identification of molecular mechanisms of sRNA-based regulation of gene expression in this emergent enteropathogen.

Show MeSH
Related in: MedlinePlus