Exploring sRNA-mediated gene silencing mechanisms using artificial small RNAs derived from a natural RNA scaffold in...

An artificial small RNA (afsRNA) scaffold was designed from an Escherichia coli sRNA, SibC. Using the lacZ reporter system, the gene silencing effects of afsRNAs were examined to explore the sRNA-mediated gene-silencing mechanisms in E. coli. Substitution of the original target recognition sequence with a new sequence recognizing lacZ mRNA led to effective reduction of lacZ 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 hfq disruption in E. coli, 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.