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. 2006 Aug;12(8):1514-20.
doi: 10.1261/rna.101606. Epub 2006 Jun 14.

Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes

Xiang Liu  1 , Feng Jiang, Savitha Kalidas, Dean Smith, Qinghua Liu
Affiliations

Dicer-2 and R2D2 coordinately bind siRNA to promote assembly of the siRISC complexes

Xiang Liu et al. RNA. 2006 Aug.

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.

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Figures

FIGURE 1.
FIGURE 1.
Generation of the r2d21 null mutant. (A, top) A schematic map of the wild-type (WT) r2d2 gene locus. Three open reading frames (ORF) were shown in the 14-kb genomic region. The P-element (EP2450) was inserted ∼0.6 kb upstream of the r2d2 ORF. (middle) A ∼4.9-kb genomic region, represented by the dash line, was deleted in the r2d21 mutant locus. The deletion was mapped by genomic PCR as illustrated below. (bottom) A 5.6-kb genomic DNA fragment that completely rescued all phenotypes of r2d21 mutant flies. (B) PCR mapping the deletion at the r2d21 mutant locus. Six (I–VI) PCR reactions (A) were performed using genomic DNA template isolated from wild-type (wt, odd lanes) or homozygous r2d21 mutant flies (r2, even lanes). The PCR condition used could not allow amplification of the ∼5.9-kb VI PCR product in the wild-type sample. (C) Lysates were prepared from wild-type flies (lane 1) and homozygous r2d21 mutant flies without (lane 2) or with the rescue transgene (lane 3). Western blots were performed with anti-R2D2 and anti-Ras antibodies.
FIGURE 2.
FIGURE 2.
Dcr-2 stabilizes R2D2 in vivo, and R2D2 is required for RNAi. (A) Lysates were prepared from wild-type (lane 1) and homozygous r2d21 (lane 2) or dcr-2R416X (lane 3) flies. The levels of Dcr-1, Dcr-2, R2D2, and Ras proteins were measured by Western blots with the corresponding antibodies (Dcr-1/Dcr-2 or R2D2/Ras used the same blot). (B) The dsRNA-processing assays were performed in buffer alone (lane 1) or with 8 μg wild-type (lane 2), r2d21 (lane 3), and dcr-2R416X (lane 4) whole fly lysates. (C) The dsRNA-initiated RISC assays were performed in buffer alone (lane 1) or 32-μg ovary lysates prepared from wild-type (lane 2), r2d21 (lane 3), dcr-2R416X (lane 4), and r2d21 mutant flies carrying the rescue transgene (lane 5).
FIGURE 3.
FIGURE 3.
Both R2D2 and Dcr-2 are required for loading siRNA onto siRISC. (A) A coomassie-stained SDS-polyacrylamide gel (PAGE) showing purified recombinant MBP-R2D2 from E. coli (lane 1) and His-tagged Dcr-2 (lane 2) or Dcr-2/R2D2 complex (lane 3) proteins from insect cells. (B) The siRNA-initiated RISC assays were performed with buffer alone (lanes 1 and 5); 32-μg wild-type (lanes 2 and 6) ovary extract; 32-μg dcr-2R416X ovary extract alone (lane 3) or supplemented with 3-pmol recombinant Dcr-2/R2D2 complex (lane 4); 32 μg r2d21 ovary extract alone (lane 7) or in combination with 12.5-pmol recombinant MBP-R2D2 (lane 8), 5-pmol Dcr-2/R2D2 complex (lane 9), and 3-pmol Dcr-2 complex (lane 10). (C) Images include a wild-type or r2d21 mutant green fluorescent embryo injected with buffer control (lanes 1 and 3), or GFP siRNAs (lanes 2 and 4). In total, 32 of 36 wild-type embryos and 0 of 40 r2d21 mutant embryos showed GFP silencing.
FIGURE 4.
FIGURE 4.
Dcr-2 and R2D2 coordinately bind siRNA duplex. The gel-shift assays were performed by incubating 5′ radiolabeled siRNA duplex and 10-pmol recombinant Dcr-2/R2D2 complex (lane 1), Dcr-2 (lane 2), MBP-R2D2 (lane 3), Dcr-2 + MBP-R2D2 (lane 4), R2D2 (lane 5), and Dcr-2 + R2D2 (lane 6). The arrowhead points to free siRNA. Two arrows refer to the Dcr-2/MBP-R2D2/siRNA and Dcr-2/R2D2/siRNA complexes, respectively.
FIGURE 5.
FIGURE 5.
Both dsRNA-binding domains (dsRBDs) of R2D2 are required for binding siRNA and promoting siRISC assembly. (A) A schematic diagram of R2D2 domain structure. The shaded boxes refer to the tandem dsRBDs of R2D2. Part of the consensus dsRBD motif was shown below. Two highly conserved alanine (A) residues were replaced by lysines (K) in the first (m1, A59–60K), second (m2, A153–154K), or both (dm, A59–60K, A153–154K) dsRBD of R2D2. (B) The poly (I): poly (C) long dsRNA-conjugated beads were used to precipitate purified wild-type (WT) and mutant MBP-R2D2 recombinant proteins. 10% input (lanes 1–4) and 100% bound proteins (lanes 5–8) were resolved by SDS-PAGE followed by coomassie staining. (C) The gel-shift assays were performed in buffer alone (lane 1); or with 3.5-pmol recombinant Dcr-2 alone (lane 2) or in combination with 12.5-pmol wild-type or various mutant MBP-R2D2 (lanes 3–6); 12.5-pmol wild-type MBP-R2D2 alone (lane 7). (D) The siRNA-initiated RISC assays were performed with buffer control (lane 1); 32 μg of wild-type ovary extract (lane 2); 32 μg r2d21 mutant extract alone (lane 3), or supplemented with 12.5-pmol wild-type or mutant MBP-R2D2 proteins (lanes 4–7).
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