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Small non-coding RNAs in Streptomyces coelicolor.

Swiercz JP - Nucleic Acids Res. (2008)

Bottom Line: We further determined the effects of growth medium composition (rich versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants.Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations.Expression of one sRNA in particular was dependent upon the sporulation-specific sigma factor sigma(WhiG).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada.

ABSTRACT
In bacteria, small RNAs (sRNAs) make important regulatory contributions to an ever increasing number of cellular processes. To expand the repertoire of known sRNAs, we sought to identify novel sRNAs in the differentiating, multicellular bacterium Streptomyces coelicolor. We describe a combined bioinformatic and experimental approach that enabled the identification and characterization of nine novel sRNAs in S. coelicolor, including a cis-encoded antisense sRNA. We examined sRNA expression throughout the S. coelicolor developmental cycle, which progresses from vegetative mycelium formation, to aerial mycelium formation and finally sporulation. We further determined the effects of growth medium composition (rich versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants. All but two of the sRNAs exhibited some degree of medium dependence, with three sRNAs being expressed exclusively during growth on one medium type. Unlike most sRNAs characterized thus far, several sRNA genes in S. coelicolor were expressed constitutively (apart from during late sporulation), suggesting a possible housekeeping role for these transcripts. Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations. Expression of one sRNA in particular was dependent upon the sporulation-specific sigma factor sigma(WhiG).

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Experimental verification of sRNAs identified using a comparative genomic search of intergenic regions. Panels on the right indicate the positioning of each sRNA (shown in black) relative to adjacent protein-coding genes (shown in grey), whilst panels on the left show northern blots probed with labelled DNA oligonucleotides (together with a 25 basepair ladder shown adjacent to scr1906). For northern blot analysis, RNA was harvested from S. coelicolor grown on either minimal or rich medium at four or five different timepoints (indicated as hours post-inoculation above each sRNA blot). 5S rRNA was used as a positive control for RNA loading and RNA integrity. For each sRNA, northern blot analysis was carried out using at least three different RNA samples to ensure the reproducibility of expression profiles.
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Figure 2: Experimental verification of sRNAs identified using a comparative genomic search of intergenic regions. Panels on the right indicate the positioning of each sRNA (shown in black) relative to adjacent protein-coding genes (shown in grey), whilst panels on the left show northern blots probed with labelled DNA oligonucleotides (together with a 25 basepair ladder shown adjacent to scr1906). For northern blot analysis, RNA was harvested from S. coelicolor grown on either minimal or rich medium at four or five different timepoints (indicated as hours post-inoculation above each sRNA blot). 5S rRNA was used as a positive control for RNA loading and RNA integrity. For each sRNA, northern blot analysis was carried out using at least three different RNA samples to ensure the reproducibility of expression profiles.

Mentions: We successfully identified transcripts corresponding to sRNAs in six different intergenic regions (Table 3 and Figure 2). The candidate regions that did not yield detectable transcripts may contain sRNA genes that are either expressed at low levels, or are simply not transcribed under the experimental conditions examined here. Alternatively, they may represent conserved DNA sequences having an as yet unknown function. Of the six identified sRNA genes, two were transcribed in a medium-specific manner: scr1906 (61–63 nt) was expressed primarily on minimal medium, while scr3261 transcripts (∼185 nt) were observed only on rich medium. In contrast, scr3045 (244 and 288 nt), scr3558 (∼67, 90 and 130 nt), scr3974 (42 and 44 nt) and scr5676 (184 nt) transcripts were observed on both minimal and rich media, although higher levels of expression were observed for scr3045 and scr3974 on rich medium, and scr5676 was more highly expressed on minimal medium. The three transcripts observed for scr3558 were examined using the ‘primer walking’ method, and were found to have a shared common core sequence, but different 5′- and 3′-ends.Table 3.


Small non-coding RNAs in Streptomyces coelicolor.

Swiercz JP - Nucleic Acids Res. (2008)

Experimental verification of sRNAs identified using a comparative genomic search of intergenic regions. Panels on the right indicate the positioning of each sRNA (shown in black) relative to adjacent protein-coding genes (shown in grey), whilst panels on the left show northern blots probed with labelled DNA oligonucleotides (together with a 25 basepair ladder shown adjacent to scr1906). For northern blot analysis, RNA was harvested from S. coelicolor grown on either minimal or rich medium at four or five different timepoints (indicated as hours post-inoculation above each sRNA blot). 5S rRNA was used as a positive control for RNA loading and RNA integrity. For each sRNA, northern blot analysis was carried out using at least three different RNA samples to ensure the reproducibility of expression profiles.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2602773&req=5

Figure 2: Experimental verification of sRNAs identified using a comparative genomic search of intergenic regions. Panels on the right indicate the positioning of each sRNA (shown in black) relative to adjacent protein-coding genes (shown in grey), whilst panels on the left show northern blots probed with labelled DNA oligonucleotides (together with a 25 basepair ladder shown adjacent to scr1906). For northern blot analysis, RNA was harvested from S. coelicolor grown on either minimal or rich medium at four or five different timepoints (indicated as hours post-inoculation above each sRNA blot). 5S rRNA was used as a positive control for RNA loading and RNA integrity. For each sRNA, northern blot analysis was carried out using at least three different RNA samples to ensure the reproducibility of expression profiles.
Mentions: We successfully identified transcripts corresponding to sRNAs in six different intergenic regions (Table 3 and Figure 2). The candidate regions that did not yield detectable transcripts may contain sRNA genes that are either expressed at low levels, or are simply not transcribed under the experimental conditions examined here. Alternatively, they may represent conserved DNA sequences having an as yet unknown function. Of the six identified sRNA genes, two were transcribed in a medium-specific manner: scr1906 (61–63 nt) was expressed primarily on minimal medium, while scr3261 transcripts (∼185 nt) were observed only on rich medium. In contrast, scr3045 (244 and 288 nt), scr3558 (∼67, 90 and 130 nt), scr3974 (42 and 44 nt) and scr5676 (184 nt) transcripts were observed on both minimal and rich media, although higher levels of expression were observed for scr3045 and scr3974 on rich medium, and scr5676 was more highly expressed on minimal medium. The three transcripts observed for scr3558 were examined using the ‘primer walking’ method, and were found to have a shared common core sequence, but different 5′- and 3′-ends.Table 3.

Bottom Line: We further determined the effects of growth medium composition (rich versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants.Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations.Expression of one sRNA in particular was dependent upon the sporulation-specific sigma factor sigma(WhiG).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada.

ABSTRACT
In bacteria, small RNAs (sRNAs) make important regulatory contributions to an ever increasing number of cellular processes. To expand the repertoire of known sRNAs, we sought to identify novel sRNAs in the differentiating, multicellular bacterium Streptomyces coelicolor. We describe a combined bioinformatic and experimental approach that enabled the identification and characterization of nine novel sRNAs in S. coelicolor, including a cis-encoded antisense sRNA. We examined sRNA expression throughout the S. coelicolor developmental cycle, which progresses from vegetative mycelium formation, to aerial mycelium formation and finally sporulation. We further determined the effects of growth medium composition (rich versus minimal medium) on sRNA gene expression, and compared wild-type sRNA expression profiles with those of four developmental mutants. All but two of the sRNAs exhibited some degree of medium dependence, with three sRNAs being expressed exclusively during growth on one medium type. Unlike most sRNAs characterized thus far, several sRNA genes in S. coelicolor were expressed constitutively (apart from during late sporulation), suggesting a possible housekeeping role for these transcripts. Others were expressed at specific developmental stages, and their expression profiles were altered in response to developmental mutations. Expression of one sRNA in particular was dependent upon the sporulation-specific sigma factor sigma(WhiG).

Show MeSH
Related in: MedlinePlus