A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila

John W. Pham, Janice L. Pellino, Young Sik Lee, Richard W. Carthew, Erik J. Sontheimer

Research output: Contribution to journalArticle

330 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)83-94
Number of pages12
JournalCell
Volume117
Issue number1
DOIs
Publication statusPublished - 2004 Apr 2

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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