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 functi...

Full description

Saved in:
Bibliographic Details
Published in:Proceedings of the National Academy of Sciences - PNAS 2002-07, Vol.99 (15), p.9942-9947
Main Authors: Yang, Dun, Buchholz, Frank, Huang, Zhongdong, Goga, Andrei, Chen, Chih-Ying, Brodsky, Frances M., Bishop, J. Michael
Format: Article
Language:English
Subjects:
Animals
Bacteria
Biological Sciences
Cell lines
Cells, Cultured
Cultured cells
DNA
Double stranded RNA
Drosophila - genetics
Endoribonucleases - metabolism
Enzymes
Escherichia coli - enzymology
Escherichia coli Proteins
Gene Silencing
Genetics
HEK293 cells
HeLa cells
Humans
Hydrolysis
Mammals
Messenger RNA
Nucleic Acid Heteroduplexes - metabolism
Ribonuclease III
Ribonucleic acid
RNA
RNA - chemical synthesis
RNA - metabolism
RNA, Double-Stranded - metabolism
RNA, Messenger - genetics
RNA, Small Interfering
RNA, Untranslated - metabolism
Small interfering RNA
Transfection
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.152327299