A Dicer-2-dependent 80s complex cleaves targeted mRNAs during RNAi in Drosophila.
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.
Pham
John W
JW
Department of Biochemistry, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.
Pellino
Janice L
JL
Lee
Young Sik
YS
Carthew
Richard W
RW
Sontheimer
Erik J
EJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D029721:Drosophila Proteins / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics*; D017665:Hydroxyl Radical / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D020365:RNA Helicases / Q000378:metabolism*; D034622:RNA Interference / Q000502:physiology*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D034743:RNA-Induced Silencing Complex / Q000096:biosynthesis* / Q000235:genetics*; D043244:Ribonuclease III; D015398:Signal Transduction / Q000235:genetics
D029721:Drosophila Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D017665:Hydroxyl Radical; D000255:Adenosine Triphosphate; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases