A role for human Dicer in pre-RISC loading of siRNAs.
RNA interference is a powerful mechanism for sequence-specific inhibition of gene expression. It is widely known that small interfering RNAs (siRNAs) targeting the same region of a target-messenger RNA can have widely different efficacies. In efforts to better understand the siRNA features that influence knockdown efficiency, we analyzed siRNA interactions with a high-molecular weight complex in whole cell extracts prepared from two different cell lines. Using biochemical tools to study the nature of the complex, our results demonstrate that the primary siRNA-binding protein in the whole cell extracts is Dicer. We find that Dicer is capable of discriminating highly functional versus poorly functional siRNAs by recognizing the presence of 2-nt 3' overhangs and the thermodynamic properties of 2-4 bp on both ends of effective siRNAs. Our results suggest a role for Dicer in pre-selection of effective siRNAs for handoff to Ago2. This initial selection is reflective of the overall silencing potential of an siRNA.
Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA.
Kumi
K
Sakurai
Mohammed
M
Amarzguioui
Dong-Ho
DH
Kim
Jessica
J
Alluin
Bret
B
Heale
Min-sun
MS
Song
Anne
A
Gatignol
Mark A
MA
Behlke
John J
JJ
Rossi
D060565:Argonaute Proteins; D002457:Cell Extracts; D015852:Eukaryotic Initiation Factor-2 / Q000502:physiology; D045325:HCT116 Cells; D057809:HEK293 Cells; D006801:Humans; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000502:physiology; D034743:RNA-Induced Silencing Complex / Q000378:metabolism*; D043244:Ribonuclease III / Q000502:physiology*; D000099253:Trans-Activation Responsive RNA-Binding Protein
D060565:Argonaute Proteins; D002457:Cell Extracts; D015852:Eukaryotic Initiation Factor-2; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D034743:RNA-Induced Silencing Complex; D043244:Ribonuclease III; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human