Research ArticleBIOMOLECULES

Bacterial antisense RNAs are mainly the product of transcriptional noise

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Science Advances  04 Mar 2016:
Vol. 2, no. 3, e1501363
DOI: 10.1126/sciadv.1501363
  • Fig. 1 Different genomic features show distinct dependency on the genomic AT content.

    The number of features was divided by the genome size for normalization and represented versus the genomic AT content. The following genomes are represented: Atu, Agrobacterium tumefaciens; Bcc, Buchnera aphidicola (str Cc); Bsu, Bacillus subtilis; Cgl, Corynebacterium glutamicum; Chl, chloroplast (Arabidopsis thaliana); Cje, Campylobacter jejuni; Eco, Escherichia coli; Hpy, Helicobacter pylori; Mge, Mycoplasma genitalium; Mhy, Mycoplasma hyopneumoniae; Mmy, Mycoplasma mycoides; Mpn, Mycoplasma pneumoniae; Mtu, Mycobacterium tuberculosis; Pau, Pseudomonas aeruginosa; Sav, Streptomyces avermitilis; Sco, Streptomyces coelicolor; Sme, Sinorhizobium meliloti; Sth, Salmonella typhimurium; Sve, Streptomyces venezuelae; Syn, Synechocystis spp., Vch, Vibrio cholerae. (A) Number of total sRNAs in different bacteria. Total sRNAs have an exponential dependency on the AT content (R2 = 0.88) and do not correlate with genome size. (B) Genome compaction (that is, number of ORFs normalized by genome size) versus AT content. Genome compaction in the different bacterial genomes analyzed shows no dependency on the AT content. Instead, the number of ORFs in bacterial genomes correlates with the genome size (R = 0.99).

  • Fig. 2 Simulation of the effect of the asRNAs, assuming that the asRNA-mRNA pairing causes duplex degradation.

    Parameters for the simulations are detailed in the Supplementary Materials. Each point of the heat maps represents the average change in the protein concentration for 100 simulations of 1000 min each, for specific parameters of asRNA and mRNA transcription rates. The remaining parameters remain constant for all the simulations. The axes represent the mRNA and asRNA concentration in the control experiments for the corresponding transcription rates scanned. (A) Changes in the mRNA concentration after 1000 min of simulation. Blue circles represent experimental data from the overexpression of asRNAs in M. pneumoniae, whereas green circles represent data from studies in Gram-negative bacteria (2931). The green ellipse delimits the region of the concentrations of most transcripts in E. coli (28). (B) Changes in the protein concentration after 1000 min of simulation. Blue circles represent experimental data from the overexpression of asRNAs in M. pneumoniae.

  • Fig. 3 Effect of the overexpression of asRNAs in their overlapping genes, measured by RNA-seq and shotgun proteomics.

    (A) Protein levels of the genes overlapping each asRNA under control conditions and in the strains transformed with the antisense constructs. Error bars represent the SD of the samples. Two of the proteins, MPN056 and MPN305, were not detected in any of the strains of M. pneumoniae. (B) mRNA levels of the genes overlapping each asRNA under control (wild-type) conditions and in the strains overexpressing the antisense transcripts. Error bars represent the SD of the samples.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/3/e1501363/DC1

    Table S1. sRNA annotation of B. aphidicola.

    Table S2. sRNA annotation of M. hyopneumoniae.

    Table S3. sRNA annotation of M. mycoides.

    Table S4. Bacterial strains used in this study.

    Table S5. asRNAs overexpressed in M. pneumoniae.

    Table S6. Shotgun proteomics results of the whole proteome of the nine clones of M. pneumoniae overexpressing asRNAs.

    Table S7. RNA-seq results of the whole transcriptome of the nine clones of M. pneumoniae overexpressing asRNAs.

    Table S8. Parameters and initial conditions used in the simulations of the asRNA effects.

    Table S9. Primers used in this study to clone the asRNAs.

    Fig. S1. Different regulatory mechanisms of sRNAs.

    Fig. S2. Theoretical and real numbers of TANAAT boxes in bacteria.

    Fig. S3. Manual annotation of sRNAs in M. hyopneumoniae.

    Fig. S4. Dependency on the AT content of different types of sRNAs.

    Fig. S5. Transcript levels of asRNAs and mRNAs in different bacteria.

    Fig. S6. Relationship between asRNAs and transcription factors in bacteria.

    Fig. S7. Simulation of the effect of the asRNAs, assuming that the pairing asRNA-mRNA causes mRNA degradation (case 2) or translation inhibition (case 3).

    References (4967)

  • Supplementary Materials

    This PDF file includes:

    • Legends for tables S1 to S3
    • Table S4. Bacterial strains used in this study.
    • Table S5. asRNAs overexpressed in M. pneumoniae.
    • Legends for tables S6 and S7
    • Table S8. Parameters and initial conditions used in the simulations of the asRNA effects.
    • Table S9. Primers used in this study to clone the asRNAs.
    • Fig. S1. Different regulatory mechanisms of sRNAs.
    • Fig. S2. Theoretical and real numbers of TANAAT boxes in bacteria.
    • Fig. S3. Manual annotation of sRNAs in M. hyopneumoniae.
    • Fig. S4. Dependency on the AT content of different types of sRNAs.
    • Fig. S5. Transcript levels of asRNAs and mRNAs in different bacteria.
    • Fig. S6. Relationship between asRNAs and transcription factors in bacteria.
    • Fig. S7. Simulation of the effect of the asRNAs, assuming that the pairing asRNA-mRNA causes mRNA degradation (case 2) or translation inhibition (case 3).
    • References (49–67)

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). sRNA annotation of B. aphidicola.
    • Table S2 (Microsoft Excel format). sRNA annotation of M. hyopneumoniae.
    • Table S3 (Microsoft Excel format). sRNA annotation of M. mycoides.
    • Table S6 (Microsoft Excel format). Shotgun proteomics results of the whole proteome of the nine clones of M. pneumoniae overexpressing asRNAs.
    • Table S7 (Microsoft Excel format). RNA-seq results of the whole transcriptome of the nine clones of M. pneumoniae overexpressing asRNAs.

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