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Park, Hongmarn,Bak, Geunu,Kim, Sun Chang,Lee, Younghoon Oxford University Press 2013 Nucleic acids research Vol.41 No.6
<P>An artificial small RNA (afsRNA) scaffold was designed from an <I>Escherichia coli</I> sRNA, SibC. Using the <I>lacZ</I> reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene-silencing mechanisms in <I>E. coli</I>. Substitution of the original target recognition sequence with a new sequence recognizing <I>lacZ</I> mRNA led to effective reduction of <I>lacZ</I> gene expression. Single-strandedness of the target recognition sequences in the scaffold was essential for effective gene silencing. The target recognition sequence was shortened to 10 nt without significant loss of gene silencing, although this minimal length was limited to a specific target mRNA sequence. In cases where afsRNAs had mismatched (forming internal loops) or unmatched (forming bulges) regions in the middle of the target recognition sequence, internal loop-forming afsRNAs were more effective in gene silencing than those that formed bulges. Unexpectedly, gene silencing by afsRNA was not decreased but increased on <I>hfq</I> disruption in <I>E. coli</I>, particularly when interactions between afsRNA and mRNA were weak, suggesting that Hfq is possibly involved in destabilization of the RNA–RNA duplex, rather than enhancement of base pairing.</P>
Systematic analysis of the role of bacterial Hfq-interacting sRNAs in the response to antibiotics
Kim, Taeyeon,Bak, Geunu,Lee, Juyeon,Kim, Kwang-sun Oxford University Press 2015 The Journal of antimicrobial chemotherapy Vol.70 No.6
<P><B>Objectives</B></P><P>To systematically analyse the interplay between the expression of Hfq-associated small non-coding RNAs (sRNAs) and antibiotic susceptibility in Gram-negative bacteria.</P><P><B>Methods</B></P><P>To identify the roles of sRNAs in the antibiotic susceptibility of <I>Escherichia coli</I> and <I>Salmonella</I> species, susceptibility tests, growth analyses and viability assays were performed using <I>E. coli</I> Hfq-associated sRNAs from overexpression libraries. Prediction, susceptibility testing of gene knockouts and expression analysis of target genes under conditions of sRNA overexpression or knockout were performed to identify candidate targets for modulating antibiotic susceptibility.</P><P><B>Results</B></P><P>The susceptibilities of <I>E. coli</I> strains overexpressing each of the 26 known Hfq-dependent sRNAs to major classes of antibiotics were determined. Induced expression of 17 sRNAs modulated the susceptibility of <I>E. coli</I> to antibiotics. Among them, four sRNA knockout strains partially or completely reversed susceptibility phenotypes of sRNA overexpression. The phenotype of OxyS, RseX or MicF was not entirely dependent on the presence of Hfq protein, in contrast to the dependency of previously characterized roles. The function of eight of nine sRNAs was found to be conserved in the response to antibiotics in <I>Salmonella</I>. Some MicF- or RyeB-mediated cellular target genes and pathways that may be important for the regulation of antibiotic susceptibility were identified. Finally, the overexpression of RyeB potentiated the efficacy of levofloxacin against MDR strains.</P><P><B>Conclusions</B></P><P>Our data indicate that Hfq-associated sRNAs potentially enable bacteria to adapt to antibiotic challenges via multifaceted approaches. Therefore, sRNA-based applications will form a new antibiotic arsenal for combating the rise in antibiotic resistance.</P>
Recognition and discrimination of target mRNAs by Sib RNAs, a <i>cis</i> -encoded sRNA family
Han, Kook,Kim, Kwang-sun,Bak, Geunu,Park, Hongmarn,Lee, Younghoon Oxford University Press 2010 Nucleic acids research Vol.38 No.17
<P>Five Sib antitoxin RNAs, members of a family of <I>cis</I>-encoded small regulatory RNAs (sRNAs) in <I>Escherichia coli</I>, repress their target mRNAs, which encode Ibs toxins. This target repression occurs only between cognate sRNA–mRNA pairs with an exception of <I>ibsA</I>. We performed co-transformation assays to assess the ability of SibC derivatives to repress <I>ibsC</I> expression, thereby revealing the regions of SibC that are essential for <I>ibsC</I> mRNA recognition. SibC has two target recognition domains, TRD1 and TRD2, which function independently. The target site for TRD1 is located within the ORF of <I>ibsC</I>, whereas the target site for TRD2 is located in the translational initiation region. The TRD1 sequence is sufficient to repress <I>ibsC</I> expression. In contrast, TRD2 requires a specific structure in addition to the recognition sequence. An <I>in vitro</I> structural probing analysis showed that the initial interactions at these two recognition sites allowed base-pairing to progress into the flanking sequences. Displacement of the TRD1 and TRD2 domains of SibC by the corresponding domains of SibD changed the target specificity of SibC from <I>ibsC</I> to <I>ibsD</I>, suggesting that these two elements modulate the cognate target recognition of each Sib RNA by discriminating among non-cognate <I>ibs</I> mRNAs.</P>