Final Activity Report Summary - RAREC (A quest for biological roles of new small RNAs in Escherichia coli)
Small noncoding RNAs have been found in all organisms ranging from bacteria (sRNA) to mammals (siRNA, miRNA), primarily as regulators of translation and message stability. The most exhaustive searches have taken place in E. coli, resulting in identification of more than 60 small RNAs. These small RNAs range from 50 to 300 nucleotides in length,are mostly encoded by intergenic regions, and are usually conserved among gram-negative bacteria.
During the functional characterisation (using proteome analysis) of the recently identified sRNAs in E. coli, we could show that overexpression of one of these, denoted MicA RNA, resulted in a strong down-regulation of OmpA protein synthesis. OmpA is one of the major outer membrane proteins in E. coli. It is known to participate in maintenance of membrane stability and to play a major role in virulence of some E. coli pathogenic strains. Thus, one can suspect that down-regulation of OmpA would be a defense mechanism against the host immune reponse. MicA RNA acts by an antisense mechanism, that is it base-pairs to its target, the ompA mRNA. My work mapped the region of base-pairing to a stretch of 16 nucleotides in the 5'-tail of MicA, and its complementarity in the 5' untranslated region of ompA mRNA. MicA interaction with this region blocks ribosome binding at the ompA translation initiation site and subsequently facilitates mRNA decay. This occurs upon entry into stationary phase when MicA becomes upregulated. Moreover, MicA RNA is also expressed in other enterobacteria such as S. typhimurium and Y. pestis, and its regulation of OmpA porin synthesis is also conserved in these pathogenic bacteria.
In a second project (to be published), I have elucidated how an SOS-related sRNA (IstR) regulates production of a toxin. Again, this system uses the antisense principle, but a conventional steric interference of the antisense RNA/target duplex with initiating ribosome appears to be ruled out. Instead, this control circuit relies on activity switches between three different, primary and processed, toxin mRNA species, and an inhibitory mechanism in which antisense binding prevents so-called "standby" ribosome binding - far away from the site at which toxin translation starts.
By analogy with miRNAs that were recently identified in eukaryotes, "in silico" searches showed that most sRNA in E. coli were antisens RNA able to regulate the expression of one or several genes (targeting different mRNAs) in a very specific manner. Moreover the target sites within genes interacting with any one given sRNA are moist often well conserved from one bacterium to another.
Results recently obtained in the host laboratory (Wagner group, ICM, Uppsala) showed that most of the sRNAs identified in E. coli are involved in the regulation of many important physiological processes in bacteria such as the envelope stress response and the SOS response to DNA damage. These results also suggest that some of these sRNAs may play a major role in the control of virulence gene expression in some pathogenic bacteria.
During the functional characterisation (using proteome analysis) of the recently identified sRNAs in E. coli, we could show that overexpression of one of these, denoted MicA RNA, resulted in a strong down-regulation of OmpA protein synthesis. OmpA is one of the major outer membrane proteins in E. coli. It is known to participate in maintenance of membrane stability and to play a major role in virulence of some E. coli pathogenic strains. Thus, one can suspect that down-regulation of OmpA would be a defense mechanism against the host immune reponse. MicA RNA acts by an antisense mechanism, that is it base-pairs to its target, the ompA mRNA. My work mapped the region of base-pairing to a stretch of 16 nucleotides in the 5'-tail of MicA, and its complementarity in the 5' untranslated region of ompA mRNA. MicA interaction with this region blocks ribosome binding at the ompA translation initiation site and subsequently facilitates mRNA decay. This occurs upon entry into stationary phase when MicA becomes upregulated. Moreover, MicA RNA is also expressed in other enterobacteria such as S. typhimurium and Y. pestis, and its regulation of OmpA porin synthesis is also conserved in these pathogenic bacteria.
In a second project (to be published), I have elucidated how an SOS-related sRNA (IstR) regulates production of a toxin. Again, this system uses the antisense principle, but a conventional steric interference of the antisense RNA/target duplex with initiating ribosome appears to be ruled out. Instead, this control circuit relies on activity switches between three different, primary and processed, toxin mRNA species, and an inhibitory mechanism in which antisense binding prevents so-called "standby" ribosome binding - far away from the site at which toxin translation starts.
By analogy with miRNAs that were recently identified in eukaryotes, "in silico" searches showed that most sRNA in E. coli were antisens RNA able to regulate the expression of one or several genes (targeting different mRNAs) in a very specific manner. Moreover the target sites within genes interacting with any one given sRNA are moist often well conserved from one bacterium to another.
Results recently obtained in the host laboratory (Wagner group, ICM, Uppsala) showed that most of the sRNAs identified in E. coli are involved in the regulation of many important physiological processes in bacteria such as the envelope stress response and the SOS response to DNA damage. These results also suggest that some of these sRNAs may play a major role in the control of virulence gene expression in some pathogenic bacteria.