ATP modulates siRNA interactions with an endogenous human Dicer complex.
Short interfering RNA (siRNA) binding by Dicer is important for RNA interference in Drosophila, but human Dicer (hDcr) has been reported to lack siRNA binding activity. We used native gel electrophoresis to characterize the siRNA-binding activity of endogenous hDcr-containing complexes in extracts from human cells. We identified a complex (D) that contains hDcr, as demonstrated by antibody supershift. Complex D appears to contain double-stranded siRNAs, and requires structural features of authentic siRNAs. Glycerol gradient sedimentation indicates that Complex D is ~250 kDa, slightly larger than hDcr alone. In addition, we found that purified recombinant hDcr (rhDcr) alone has siRNA binding activity. Complex D migrates more slowly than the rhDcr/siRNA complex in a native gel, suggesting that it contains at least one additional factor. hDcr directly contacts siRNAs within Complex D, as indicated by crosslinking. The endogenous complex is significantly enhanced by ATP, unlike the siRNA-binding activity of purified rhDcr, suggesting the existence of additional factors that can enforce the ATP dependence of endogenous hDcr/siRNA interactions. Complex D could impinge upon the RISC assembly pathway in humans, similar to an analogous complex in Drosophila.
Pellino
Janice L
JL
Department of Biochemistry, Molecular Biology, and Cell Biology, Northwestern University, Evanston, IL 60208-3500, USA.
Jaskiewicz
Lukasz
L
Filipowicz
Witold
W
Sontheimer
Erik J
EJ
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D003432:Cross-Linking Reagents; D053487:DEAD-box RNA Helicases; D004722:Endoribonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007668:Kidney / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043244:Ribonuclease III
D003432:Cross-Linking Reagents; D034741:RNA, Small Interfering; D000255:Adenosine Triphosphate; D004722:Endoribonucleases; C408087:DICER1 protein, human; D043244:Ribonuclease III; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases