Results: 5

1.
Fig. 2.

Fig. 2. From: Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

SmD1 interacts with components of the siRNA biogenesis machinery. (A) Lysates from S2 cells expressing Flag-tagged SmD1 or control proteins (U1-70K or Ran) were immunoprecipitated with anti-Flag antibody. Identical copies of input and immunoprecipitated material were subject to immunoblotting using various antibodies (left). (B) Total RNA was extracted from immunopurified SmD1 complexes and subject to RT-qPCR to measure levels of CG4068 (n = 4) or the control mRNA rp49. Samples recovered from a parallel immunoprecipitation using nonimmune serum (n. i.) serve as negative control. (C and D) Total RNA was extracted from immunopurified TAP-tagged Dcr-2 or SmD1 complexes and subject to RT-qPCR to measure levels of the esi-2.1 precursor transcript CG4068 (C) or the control mRNA rp49 (D). Fold enrichment (n = 3) compared with control samples (from cells expressing the TAP tag only) is shown on a log scale. (E) S2 cells stably expressing TAP–Dcr-2 were treated with control (Luc) or SmD1 dsRNAs. Relative enrichment of CG4068 in purified TAP–Dcr-2 complexes was measured by RT-qPCR (n = 3). Data are shown as mean + SD in E or mean + SEM in B, C, and D. **P < 0.01.

Xiao-Peng Xiong, et al. Proc Natl Acad Sci U S A. 2013 October 8;110(41):16520-16525.
2.
Fig. 3.

Fig. 3. From: Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

Loss of SmD1 abolishes siRISC assembly. (A) Cytoplasmic lysates from various knockdown cells (Top) were incubated with a synthetic siRNA duplex. The slicer activity of the resulting siRISC was measured as cleavage of the cap-radiolabeled target mRNA. (B) The slicer activity of the siRISC assembled in lysates from various knockdown cells (Bottom) was quantified (n ≥ 3). (C) Minimal RISC was immunopurified from Flag–AGO2 cell lysates using Flag-agarose beads. Purified minimal RISC was then incubated with lysates from control or SmD1-knockdown cells in the presence of an siRNA duplex radiolabeled on the guide strand. AGO2-bound RNAs were extracted from the beads and resolved by denaturing urea-PAGE. (D) siRNA duplex loading was quantified as the amount of radiolabeled siRNA guide strand recovered from the AGO2 siRISC assembled in lysates from various knockdown cells (n = 3). (E) siRNA duplex unwinding was measured as in C except that an excess of unlabeled guide siRNA strand was added to the reaction and AGO2-associated RNAs were resolved by native PAGE to separate ds-siRNAs and ss-siRNAs. The unwinding index was calculated and is shown at the bottom. A representative image from two independent experiments is shown. (F) Passenger-strand nicking was measured as described in C except that the siRNA duplex was radiolabeled on the passenger strand and an excess of 2′-O-methylated oligo complementary to the passenger strand was added. RNAs were extracted from the reaction mixture and resolved by denaturing urea-PAGE. (G) The passenger-strand nicking activity of lysates from various knockdown cells (Bottom) was quantified. Data are shown as mean + SEM in B, D, and G. ***P < 0.001; n.s., nonsignificant.

Xiao-Peng Xiong, et al. Proc Natl Acad Sci U S A. 2013 October 8;110(41):16520-16525.
3.
Fig. 4.

Fig. 4. From: Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

SmD1 interacts with siRISC components. (A) Flag–SmD1 was coexpressed with various T7-tagged proteins (Top) in S2 cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The isolated complexes were treated with or without RNase A then analyzed by immunoblotting with anti-Flag and anti-T7 antibodies. (B) Flag–SmD1 complexes were immunopurified from S2 cells, treated with or without RNase, and analyzed by immunoblotting with antibodies against AGO2, dFMR, Bel, or Flag. Asterisks in A and B indicate nonspecific bands. (C) Flag–SmD1 from lysates of stably transfected cells was immobilized onto anti-Flag agarose beads and then incubated with buffer or lysates from naïve S2 cells. RISC assembly was assessed by measuring slicer activity associated with the isolated beads (lanes 1 and 2). (D) Cell lysates were subject to slicer activity assays (Upper) before (lane 1) or after (lane 2) immunodepletion of Flag–SmD1 using anti-Flag agarose beads. AGO2 protein levels in aliquots of lysates were analyzed by immunoblotting (Lower). Control cell lysates were subject to a similar immunodepletion procedure using nonimmune (n. i.) serum (lanes 3 and 4). (E) Lysates from S2 cells expressing various Flag-tagged proteins (Top) were immunoprecipitated with anti-Flag antibody. Associated RNAs were extracted and analyzed by Northern blotting to detect esi-2.1 and miR-2b. Of note, expression of Flag–AGO2 reduced levels of total AGO2 (possibly owing to cosuppression) (45) and esi-2.1 in the input sample.

Xiao-Peng Xiong, et al. Proc Natl Acad Sci U S A. 2013 October 8;110(41):16520-16525.
4.
Fig. 1.

Fig. 1. From: Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

Loss of SmD1 compromises RNAi and siRNA biogenesis. (A) S2 cells were transfected with Dcr-2, SmD1, or control (LacZ) dsRNA together with expression constructs for a Renilla luciferase reporter containing complementary binding sites for esi-2.1 and a firefly luciferase control reporter. Loss of RNAi was measured as derepression of the luciferase reporter activity (n ≥ 3). (B) S2 cells treated with various dsRNAs (below) were infected with DCV. DCV RNA levels were measured by RT-qPCR (n ≥ 3). (C) Northern blotting to measure levels of the endogenous siRNA esi-2.1 or 2S rRNA (loading control) in various dsRNA-treated S2 cells. (D) Quantification of esi-2.1 levels (n = 4) normalized against 2S rRNA levels and compared with controls. (E) Northern blotting to measure esi-1.1 and 2S rRNA levels in various knockdown cells. (F) Quantification of esi-1.1 levels. Dcr-2 and Drosha, n = 2; SmD1, n = 4. (G) Cytoplasmic lysates from S2 cells treated with various dsRNAs were incubated with radiolabeled dsRNA substrate to generate 21-nt siRNAs. RNAs were extracted and resolved by denaturing urea-PAGE. (H) The amount of siRNAs is quantified and normalized to controls (n = 3). (I) Kinetic analysis of Dcr-2 activity using lysates from control or SmD1 knockdown cells. Fraction of dsRNA substrate processed by Dcr-2 at various time points is shown. Data are shown as mean + SD in A, D, F, and H or mean + SEM in B. *P < 0.05, **P < 0.01, ***P < 0.001. Unless noted otherwise, dsRNAs against Renilla luciferase or firefly luciferase serve as controls.

Xiao-Peng Xiong, et al. Proc Natl Acad Sci U S A. 2013 October 8;110(41):16520-16525.
5.
Fig. 5.

Fig. 5. From: Core small nuclear ribonucleoprotein particle splicing factor SmD1 modulates RNA interference in Drosophila.

RNAi and splicing machineries are physically and functionally distinct. (A) S2 cells were treated with various dsRNAs. Levels of esi-2.1 and the control 2S rRNA were measured by Northern blotting. (B and C) Cytoplasmic lysates were assayed for (B) dsRNA processing by dicer assays and (C) mRNA cleavage by slicer assays. In AC, relative levels of the indicated product are shown beneath the lanes. Representative images from two independent experiments are shown. (D) Splicing activity was assessed in various knockdown cells (Top) by examining splicing patterns of the indicated mRNAs (Bottom) by RT-PCR. The splice variants are shown on the right. The asterisk indicates an unannotated splice variant. (E) Lysates from S2 cells expressing various Flag-tagged proteins were immunoprecipitated using anti-Flag antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against AGO2 and Flag. (F and G) Total RNA was extracted from immunopurified SmD1 (F) or Flag–AGO2 complexes (G). Levels of various snRNAs or the control mRNAs mus308 and rp49 were measured by RT-qPCR. Results are the mean + SEM of n = 4; *P < 0.05. Samples recovered from a parallel immunoprecipitation using nonimmune serum (n. i.) serve as negative control. (H) A schematic depicting the proposed roles of SmD1 in the initiation (siRNA biogenesis) and effector (siRISC assembly and/or mRNA cleavage) steps of RNAi.

Xiao-Peng Xiong, et al. Proc Natl Acad Sci U S A. 2013 October 8;110(41):16520-16525.

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