RESUMO
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.
Assuntos
Trifosfato de Adenosina/farmacologia , Endorribonucleases/metabolismo , RNA Helicases/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Reagentes de Ligações Cruzadas , RNA Helicases DEAD-box , Células HeLa , Humanos , Rim/metabolismo , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ribonuclease IIIRESUMO
We use native gel electrophoresis to characterize complexes that mediate RNA interference (RNAi) in Drosophila. Our data reveal three distinct complexes (R1, R2, and R3) that assemble on short interfering RNAs (siRNAs) in vitro. To form, all three complexes require Dicer-2 (Dcr-2), which directly contacts siRNAs in the ATP-independent R1 complex. R1 serves as a precursor to both the R2 and R3 complexes. R3 is a large (80S), ATP-enhanced complex that contains unwound siRNAs, cofractionates with known RNAi factors, and binds and cleaves targeted mRNAs in a cognate-siRNA-dependent manner. Our results establish an ordered biochemical pathway for RISC assembly and indicate that siRNAs must first interact with Dcr-2 to reach the R3 "holo-RISC" complex. Dcr-2 does not simply transfer siRNAs to a distinct effector complex, but rather assembles into RISC along with the siRNAs, indicating that its role extends beyond the initiation phase of RNAi.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Helicases/metabolismo , Interferência de RNA/fisiologia , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Complexo de Inativação Induzido por RNA/biossíntese , Complexo de Inativação Induzido por RNA/genética , Trifosfato de Adenosina/metabolismo , Animais , Sítios de Ligação/genética , Radical Hidroxila/metabolismo , Substâncias Macromoleculares , Peso Molecular , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Interferente Pequeno/química , RNA Interferente Pequeno/genética , Ribonuclease III , Transdução de Sinais/genéticaRESUMO
During RNA interference (RNAi), Dicer generates short interfering RNAs (siRNAs), which then guide target mRNA cleavage by the RISC complex. Now, Liu et al. identify R2D2, a Dicer-associated protein that is important for siRNA incorporation into RISC, thus linking the initiation and execution phases of RNAi.
