A genome-wide RNAi screen reveals a role for Ge-1 in miRNA-mediated gene silencing. (A) S2 cells were transfected with a mixture of plasmids containing the F-Luc-CG10011 reporter, a plasmid expressing miR-12 (+miR-12) or the corresponding empty vector (−miR-12), and a plasmid expressing Renilla luciferase (R-Luc). Firefly luciferase activity (black bars) and mRNA levels (gray bars) are normalized to that of the Renilla luciferase and set to 100 in cells transfected with the empty vector. Mean values ± standard deviations from three independent experiments are shown. (B) The results of the RNAi screen are represented as z-scores of two independent screens (see Materials and Methods). (Blue dots) Individual dsRNAs in the library; (red diamonds) components of the miRNA pathway; (green dot) Ge-1 (CG6181); (dashed red line) cutoff used (average z-score > 10). (C–E) S2 cells were treated with the indicated dsRNAs on days 0 and 4. On day 6, cells were transfected with the mixture of plasmids described in A. (C) The effectiveness of the depletions was analyzed by Western blotting, with the antibodies indicated on the left. In lanes 1–4, dilutions of untreated cells are loaded. Anti-tubulin antibodies were used as a loading control. Numbers above the lanes indicate the regions targeted by the dsRNAs and correspond to amino acids in the Ge-1 protein sequence. (D) Firefly luciferase activity (black bars) and mRNA levels (gray bars) are normalized to that of the Renilla luciferase. Furthermore, for each knockdown, the normalized values of F-Luc activity and mRNA levels in the presence of the miRNA are divided by those obtained in the absence of the miRNA. These ratios are set to unity in cells treated with GFP dsRNA. Mean values ± standard deviations from three independent experiments are shown. (E) Northern blot analysis of representative RNA samples isolated from S2 cells shown in D.

Ge-1 is a conserved component of metazoan P-bodies. (A) Domain architecture of Ge-1. (Dark-green triangles) Canonical WD40 repeats; (light-green triangles) divergent WD40 repeats; (yellow box) flexible linker rich in serines; (dark-blue box) C-terminal conserved domain; (cyan box) highly conserved motif. Numbers below the protein outline represent amino acid positions at fragment boundaries for the D. melanogaster protein. The protein domains sufficient for the localization to P-bodies are shown in red. (B) Confocal fluorescent micrograph of S2 cells expressing HA fusions of the Ge-1 domains indicated on the left. Cells were stained with anti-HA and affinity-purified anti-Tral antibodies. Bar, 5 μm.

Expression profiles of D. melanogaster S2 cells depleted of AGO1 or decapping activators. (A) S2 cells were treated with the dsRNAs indicated below the lanes. The number (nb) of independent expression profiles obtained per depleted protein is indicated in brackets. Average expression levels of transcripts detectable in all profiles (5899 mRNAs) are shown. RNAs are represented as lines colored relative to their expression levels, as indicated on the left. Numbers above the lanes indicate rank correlation coefficients relative to AGO1, DCP1, or EDC3 as indicated. The experiment tree was calculated using the distance option in the GeneSpring software (Euclidean distance). (B) RNAs at least 1.5-fold overrepresented in the three independent profiles obtained for AGO1, and <1.5-fold up-regulated in at least nine of 10 profiles of decapping activators. (C) RNAs at least 1.5-fold overrepresented in the two profiles obtained for two or more decapping activators, and <1.5 up-regulated in the AGO1 knockdown. (D) mRNAs at least 1.5-fold overrepresented in the three independent profiles obtained for AGO1, and in the two profiles obtained for at least two decapping activators. (E) RNAs at least 1.5-fold overrepresented in the three independent profiles obtained for AGO1, and in five of six profiles obtained for DCP-1, Ge-1, and HPat. (F) RNAs at least 1.5-fold overrepresented in the three independent profiles obtained for AGO1 and in all profiles obtained for EDC3 and LSm1. Note that the lines representing individual mRNAs are compressed in A. The number of mRNAs displayed per panel is indicated on the left.

Decapping activators suppress silencing of miRNA targets regulated at the mRNA level. (A–D) S2 cells were treated with the indicated dsRNAs on days 0 and 4. On day 6, cells were transfected with a mixture of plasmids as described in Figure 4. Firefly luciferase activity and mRNA levels are analyzed as described in Figure 4. (B,D) Northern blot analysis of representative RNA samples isolated from S2 cells shown in A and C, respectively.

Decapping activators suppress silencing of miRNA targets regulated at the mRNA levels. (A–D) S2 cells were treated with the indicated dsRNAs on days 0 and 4. On day 6, cells were transfected with a mixture of plasmids as described in Figure 4. Samples are analyzed as described in Figure 4. (B,D) Northern blot analysis of representative RNA samples isolated from S2 cells shown in A and C, respectively.

Decapping activators have partially redundant functions in silencing. (A–D) S2 cells were treated with the indicated dsRNAs on days 0 and 4. On day 6, cells were cotransfected with a mixture of plasmids expressing firefly luciferase (F-Luc) followed by the indicated 3′ UTRs, plasmids expressing miRNA primary transcripts (+miR-12) or the corresponding empty vector (−miR-12), and a plasmid expressing Renilla luciferase (R-Luc). (A,C) Firefly luciferase activity and mRNA levels are normalized to that of the Renilla luciferase. For each knockdown, the normalized values of F-Luc activity and mRNA levels are set to 100 in cells transfected with the empty vector (not shown except for control cells). Mean values ± standard deviations from three independent experiments are shown. (B,D) Northern blot analysis of representative RNA samples isolated from S2 cells shown in A and C, respectively.

Decapping activators suppress silencing of miR-1 targets. (A–D) S2 cells were treated with the indicated dsRNAs on days 0 and 4. On day 6, cells were transfected with a mixture of plasmids as described in Figure 4. Firefly luciferase activity and mRNA levels are analyzed as described in Figure 4. (B,D) Northern blot analysis of representative RNA samples isolated from S2 cells shown in A and C, respectively.

miRNAs elicit mRNA decay via translation-dependent and translation-independent mechanisms. (A–G) S2 cells were cotransfected with a mixture of plasmids as described in Figure 4. Cells were treated with cycloheximide (CHX) as indicated. Firefly luciferase mRNA levels are normalized to that of the Renilla luciferase. The normalized values of F-Luc mRNA are set to 100 in cells transfected with the empty vector (i.e., in the absence of the miRNA; black bars). Mean values ± standard deviations from three independent experiments are shown.