Nucleic Acids Research Advance Access published online on May 27, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp447
Methods online |
Specific gene silencing by artificial trans-encoded small noncoding RNAs in bacteria
Department of Molecular Biology, School of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, People's Republic of China
*To whom correspondence should be addressed. Tel: +86 532 82031680; Fax: +86 532 82033054; Email: yuwg66{at}ouc.edu.cn
Received February 16, 2009. Revised May 9, 2009. Accepted May 12, 2009.
Recently, numerous small noncoding RNAs (sRNAs) with important regulatory roles have been identified in bacteria. 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 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 natural bacterial trans-encoded sRNAs, we developed the designing principle of atsRNA. Most of the atsRNAs effectively suppressed the expression of exogenous EGFP gene and endogenous uidA gene in Escherichia coli. Further studies demonstrated that the mRNA base-pairing region and AU rich Hfq binding site were crucial for the activity of atsRNA. The atsRNA-mediated gene silencing was Hfq dependent. atsRNA led to translational repression and RNase-E-dependent degradation of target mRNA, and the translation inhibition was the primary event for gene silencing. Our findings demonstrated that atsRNA was an effective RNA tool for specific gene silencing in bacteria.