Abstract
In Drosophila melanogaster, the Dicer-2/R2D2 complex initiates RNA interference (RNAi) by processing long double-stranded RNA (dsRNA) into small interfering RNA (siRNA). Recent biochemical studies suggest that the Dcr-2/R2D2 complex also facilitates incorporation of siRNA into the RNA-induced silencing complex (siRISC). Here we present genetic evidence that R2D2 and Dcr-2 are both required for loading siRNA onto the siRISC complex. Consistent with this, only the Dcr-2/R2D2 complex, but neither Dcr-2 nor R2D2 alone, can efficiently interact with duplex siRNA. Furthermore, both dsRNA-binding domains of R2D2 are critical for binding to siRNA and promoting assembly of the siRISC complexes.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Drosophila Proteins / analysis
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Drosophila Proteins / chemistry
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Drosophila Proteins / genetics
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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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Histidine / chemistry
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Homozygote
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Insect Proteins / metabolism*
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Open Reading Frames
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Protein Structure, Tertiary
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RNA Helicases / analysis
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RNA Helicases / metabolism*
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RNA, Double-Stranded / metabolism
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RNA, Small Interfering / biosynthesis
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RNA, Small Interfering / metabolism*
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RNA-Binding Proteins / analysis
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RNA-Binding Proteins / chemistry
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RNA-Binding Proteins / genetics
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RNA-Binding Proteins / metabolism*
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RNA-Induced Silencing Complex / metabolism*
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Recombinant Proteins / chemistry
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Recombinant Proteins / isolation & purification
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Recombinant Proteins / metabolism
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Ribonuclease III
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Sequence Deletion
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Thermodynamics
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Transgenes
Substances
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Drosophila Proteins
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Insect Proteins
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R2D2 protein, Drosophila
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RNA, Double-Stranded
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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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Histidine
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DCR-2 protein, Drosophila
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Ribonuclease III
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RNA Helicases