Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells.
Small interfering RNA (siRNA) has become a powerful tool for selectively silencing gene expression in cultured mammalian cells. Because different siRNAs of the same gene have variable silencing capacities, RNA interference with synthetic siRNA is inefficient and cost intensive, especially for functional genomic studies. Here we report the use of Escherichia coli RNase III to cleave double-stranded RNA (dsRNA) into endoribonuclease-prepared siRNA (esiRNA) that can target multiple sites within an mRNA. esiRNA recapitulates the potent and specific inhibition by long dsRNA in Drosophila S2 cells. In contrast to long dsRNA, esiRNA mediates effective RNA interference without apparent nonspecific effect in cultured mammalian cells. We found that sequence-specific interference by esiRNA and the nonspecific IFN response activated by long dsRNA are independent pathways in mammalian cells. esiRNA works by eliciting the destruction of its cognate mRNA. Because of its simplicity and potency, this approach is useful for analysis of mammalian gene functions.
Yang
Dun
D
G. W. Hooper Foundation and Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0552, USA. dyang20@itsa.ucsf.edu
Buchholz
Frank
F
Huang
Zhongdong
Z
Goga
Andrei
A
Chen
Chih-Ying
CY
Brodsky
Frances M
FM
Bishop
J Michael
JM
D000818:Animals; D002478:Cells, Cultured; D004330:Drosophila / Q000235:genetics; D004722:Endoribonucleases / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D020868:Gene Silencing; D006801:Humans; D006868:Hydrolysis; D008322:Mammals; D009692:Nucleic Acid Heteroduplexes / Q000378:metabolism*; D012313:RNA / Q000138:chemical synthesis / Q000378:metabolism*; D012330:RNA, Double-Stranded / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D034741:RNA, Small Interfering; D022661:RNA, Untranslated / Q000378:metabolism*; D043244:Ribonuclease III
D029968:Escherichia coli Proteins; D009692:Nucleic Acid Heteroduplexes; D012330:RNA, Double-Stranded; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D022661:RNA, Untranslated; D012313:RNA; D004722:Endoribonucleases; D043244:Ribonuclease III; C468366:ribonuclease III, E coli