Abstract
The RNA interference (RNAi) pathway is initiated by processing long double-stranded RNA into small interfering RNA (siRNA). The siRNA-generating enzyme was purified from Drosophila S2cells and consists of two stoichiometric subunits: Dicer-2(DCR-2) and a previously unknown protein that we named R2D2. R2D2 is homologous to the Caenorhabditis elegans RNAi protein RDE-4. Association with R2D2 does not affect the enzymatic activity of DCR-2. Rather, the DCR-2/R2D2 complex, but not DCR-2 alone, binds to siRNA and enhances sequence-specific messenger RNA degradation mediated by the RNA-initiated silencing complex (RISC). These results indicate that R2D2 bridges the initiation and effector steps of the Drosophila RNAi pathway by facilitating siRNA passage from Dicer to RISC.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Amino Acid Sequence
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Animals
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Argonaute Proteins
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Biotinylation
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Caenorhabditis elegans / genetics
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Caenorhabditis elegans / metabolism
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Caenorhabditis elegans Proteins / chemistry
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Cell Line
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Chemical Precipitation
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Drosophila Proteins / chemistry
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Drosophila Proteins / genetics
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Drosophila Proteins / isolation & purification*
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Drosophila Proteins / metabolism*
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Drosophila melanogaster / genetics*
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Drosophila melanogaster / metabolism
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Electrophoretic Mobility Shift Assay
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Endoribonucleases / genetics
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Endoribonucleases / isolation & purification
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Endoribonucleases / metabolism*
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Kinetics
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Molecular Sequence Data
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Mutation
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Protein Structure, Tertiary
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RNA Helicases / genetics
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RNA Helicases / isolation & purification*
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RNA Helicases / metabolism*
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RNA Interference*
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RNA, Double-Stranded / metabolism
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RNA, Messenger / metabolism
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RNA, Small Interfering / metabolism*
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / isolation & purification
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RNA-Binding Proteins / metabolism*
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RNA-Induced Silencing Complex / isolation & purification
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RNA-Induced Silencing Complex / metabolism
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Recombinant Proteins / metabolism
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Ribonuclease III
Substances
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AGO2 protein, Drosophila
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Argonaute Proteins
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Caenorhabditis elegans Proteins
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Drosophila Proteins
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R2D2 protein, Drosophila
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RDE-4 protein, C elegans
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RNA, Double-Stranded
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RNA, Messenger
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RNA, Small Interfering
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RNA-Binding Proteins
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RNA-Induced Silencing Complex
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Recombinant Proteins
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DCR-2 protein, Drosophila
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Endoribonucleases
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Ribonuclease III
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RNA Helicases