Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

doi:10.1073/pnas.152327299

Short RNA duplexes produced by hydrolysis with Escherichia coli RNase III mediate effective RNA interference in mammalian cells

^*G. W. Hooper Foundation and Department of Microbiology and Immunology, and ^§Division of Hematology/Oncology, Department of Medicine, University of California, San Francisco, CA 94143-0552

Contributed by J. Michael Bishop

Abstract

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.

Footnotes

↵ † To whom reprint requests should be addressed. E-mail: dyang20{at}itsa.ucsf.edu.
↵ ‡ Present address: Max Planck Institute–Molecular Cell Biology and Genetics, Pfotenhauer Strasse 108, 01307 Dresden, Germany.
Abbreviations:

RNAi,

RNA interference;

dsRNA,

double-stranded RNA;

siRNA,

small interfering RNA;

esiRNA,

endoribonuclease-prepared siRNA;

F-luc,

firefly luciferase;

R-luc,

Renilla luciferase;

GST,

glutathione S-transferase;

RPE,

retinal pigment epithelial;

LCa/LCb,

clathrin light chain a/b