Specific Gene Silencing By Artificial Trans-encoded SRNA In Bacterial

Small noncoding RNAs (sRNAs) are widespread in both eukaryotes and prokaryotes. These small noncoding RNAs, which function as regulators of gene expression, constitute a structurally diverse class of molecules that are typically shorter than 400 nucleotides (nt) in length and do not contain expressed open reading frames (ORFs). The eukaryotic small noncoding RNAs, such as microRNA (miRNA), short interfering RNA (siRNA) and Piwi-interacting RNA (piRNA) have making a splash during the past few years. Recently, with the development of the experimental and computational approaches, hundreds of sRNAs have been identified in prokaryotes, especially in bacterial. As their eukaryotic counterparts, a major class of bacterial trans-encoded sRNAs acts by basepairing with target mRNAs, resulting in changes in translation and stability of the mRNA. RNA interference (RNAi) has become an extraordinarily powerful RNA silencing tool for elucidating and manipulating gene functions in eukaryotes. However, such an effective RNA silencing tool remains to be developed for prokaryotes in which homologous recombination and transposon mutagenesis remain to be the major tools of deciphering the function of genes.In this study, we described firstly the use of artificial trans-encoded sRNAs (atsRNAs) for specific gene silencing in bacteria. Based on the common structural characteristics of the natural bacterial sRNAs, we have developed the principle and process for atsRNA design. atsRNA was designed to be a modular structure composed of three elements: mRNA basepairing region, Hfq binding site and typical Rho-independent terminator. The three component parts were selected randomly and then assembled into a series of atsRNA candidates whose secondary structures were then predicted by MFOLD program. atsRNAs should be selected from the atsRNA candidates according to predicted secondary structure and certain other selecting criteria. The complementary DNA oligonucleotides corresponding to the designed atsRNAs and cloned into a plasmid vector under the control of tac promoter for expression of atsRNAs. To evaluate the feasibility of this method, an exogenous EGFP gene on a multi-copy plasmid and an endogenous uidA gene encoding beta-glucuronidase were used as targets for the atsRNAs. Most, if not all, atsRNAs inhibited the expression of the target genes to effectively when they were expressed by IPTG addition in E. coli.Further studies demonstrated that the secondary structure (e.g. stem-loop structure of mRNA basepairing region, the number of unpaired nucleotides in a loop structure) and stability were crucial for the activity of atsRNA. Additionally, mutations in either mRNA basepairing regions or Hfq binding sites abolished the ability to repress the expression of target genes. This result also indicated that atsRNA acted by basepairing with target mRNA. The arsRNA-mediated gene silencing was Hfq dependent and Hfq could stabilize the atsRNA by binding to the atsRNA directing. atsRNA led to translational repression and RNase E dependent degradation of target mRNA, and the translation inhibition was the primary event for gene silencing. As for certain natural regulatory RNAs, degradation of the mRNA does not contribute to the efficiency of repression.Finally, in order to substantiate our findings, we generated atsRNAs for silencing of three essential genes murA, trmA and ygjD. We succeeded to cause the growth inhibition of E. coli cells by expressing atsRNAs complementary to several essential genes, suggesting that these atsRNAs inhibit efficiently the expression of these essential genes. All together, our findings demonstrated that atsRNA was an effective RNA tool for specific gene silencing in bacteria.Recent studies have demonstrated that numerous endogenous trans-encoded sRNAs have crucial roles in bacterial stress responses and virulence regulation. Therefore, atsRNAs targeting against virulence genes would function effectively in bacterial pathogens and it could potentially serve as antibiotics. Given the emergence and increasing prevalence of bacterial strains that are resistant to available antibiotics, atsRNAs will provide an alternative approach to antimicrobial therapy that offers promising opportunities to inhibit pathogenesis and its consequences without placing immediate life-or-death pressure on the target bacterium.