Coexpression of Argonaute-2 enhances RNA interference toward perfect match binding sites.
RNAi is widely applied to inhibit expression of specific genes, but it is limited by variable efficiency and specificity of empirically designed siRNA or shRNA constructs. This complicates studies targeting individual genes and significantly impairs large-scale screens using genome-wide knockdown libraries. Here, we show that ectopic expression of the RISC slicer Argonaute-2 (Ago2, eIF2C2) dramatically enhances RNAi specifically for mRNA targets with perfectly matched binding sites. This effect depends on its endonuclease activity and is uncoupled from its regulation of microRNA expression. To model the application of Ago2 coexpression with shRNA knockdown, we targeted the EGF receptor (EGFR) in lung cancer cells exhibiting oncogene addiction to EGFR. Whereas multiple empirically designed shRNA constructs exhibited highly divergent efficiencies in mediating EGFR knockdown and cell killing, coexpression of Ago2 resulted in uniform and highly specific target gene suppression and apoptosis in EGFR-dependent cells. Codelivery of Ago2 with shRNA constructs or siRNA duplexes thus provides a strategy to enhance the efficacy and the specificity of RNAi in experimental and potentially therapeutic settings.
Diederichs
Sven
S
Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129-2020, USA. s.diederichs@dkfz.de
Jung
Stephanie
S
Rothenberg
S Michael
SM
Smolen
Gromoslaw A
GA
Mlody
Barbara G
BG
Haber
Daniel A
DA
D060565:Argonaute Proteins; D001665:Binding Sites; D016923:Cell Death; D002460:Cell Line; D066246:ErbB Receptors / Q000378:metabolism; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism
C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D034741:RNA, Small Interfering; D066246:ErbB Receptors