@article{2695843092a34e45bec680a71e4b1e98,
title = "A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila",
abstract = "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.",
author = "Pham, {John W.} and Pellino, {Janice L.} and Lee, {Young Sik} and Carthew, {Richard W.} and Sontheimer, {Erik J.}",
note = "Funding Information: We are very grateful to P. Zamore for the guanylyl transferase that we used in our early experiments; A. Caudy, G. Hannon, and E. Izaurralde for antibodies; and Q. Liu and X. Wang for antibodies and for communicating results before publication. We thank J. Gorra and Z. He for help with fly maintenance and lysate preparation, R. Lieberman for assistance with column chromatography, T. Karginov and R. Fahlman for help with sedimentation analysis, and P. Bellare, A. Lamond, J. Staley, and O. Uhlenbeck for comments on the manuscript. J.W.P. and J.L.P. were supported by NIH Training Grants in Biophysics and the Molecular and Cellular Basis of Disease, respectively. Y.S.L. was supported by a FRAXA postdoctoral fellowship. This work was supported by a Burroughs Wellcome Fund New Investigator Award to E.J.S. and by a grant from the National Institutes of Health (GM068743-01) to R.W.C. and E.J.S. ",
year = "2004",
month = apr,
day = "2",
doi = "10.1016/S0092-8674(04)00258-2",
language = "English (US)",
volume = "117",
pages = "83--94",
journal = "Cell",
issn = "0092-8674",
publisher = "Cell Press",
number = "1",
}