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.
Sakurai
Kumi
K
Department of Molecular and Cellular Biology, Beckman Research Institute, City of Hope, 1450 East Duarte Road, Duarte, CA 91010, USA.
Amarzguioui
Mohammed
M
Kim
Dong-Ho
DH
Alluin
Jessica
J
Heale
Bret
B
Song
Min-sun
MS
Gatignol
Anne
A
Behlke
Mark A
MA
Rossi
John J
JJ
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