Molecular requirements for RNA-induced silencing complex assembly in the Drosophila RNA interference pathway.
Complexes in the Drosophila RNA-induced silencing complex (RISC) assembly pathway can be resolved using native gel electrophoresis, revealing an initiator called R1, an intermediate called R2, and an effector called R3 (now referred to as holo-RISC). Here we show that R1 forms when the Dicer-2/R2D2 heterodimer binds short interfering RNA (siRNA) duplexes. The heterodimer alone can initiate RISC assembly, indicating that other factors are dispensable for initiation. During assembly, R2 requires Argonaute 2 to convert into holo-RISC. This requirement is reminiscent of the RISC-loading complex, which also requires Argonaute 2 for assembly into RISC. We have compared R2 to the RISC-loading complex and show that the two complexes are similar in their sensitivities to ATP and to chemical modifications on siRNA duplexes, indicating that they are likely to be identical. We have examined the requirements for RISC formation and show that the siRNA 5'-termini are repeatedly monitored during RISC assembly, first by the Dcr-2/R2D2 heterodimer and again after R2 formation, before siRNA unwinding. The 2'-position of the 5'-terminal nucleotide also affects RISC assembly, because an siRNA strand bearing a 2'-deoxyribose at this position can inhibit the cognate strand from entering holo-RISC; in contrast, the 2'-deoxyribose-modified strand has enhanced activity in the RNA interference pathway.
Pham
John W
JW
Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illinois 60208-3500, USA.
Sontheimer
Erik J
EJ
D000818:Animals; D060565:Argonaute Proteins; D019281:Dimerization; D004330:Drosophila / Q000235:genetics* / Q000378:metabolism*; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008954:Models, Biological; D046912:Multiprotein Complexes; D009690:Nucleic Acid Conformation; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D034743:RNA-Induced Silencing Complex / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III
C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex; D011994:Recombinant Proteins; C478069:DCR-2 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases