1. Drosophila Argonaute1 and Argonaute2 Employ Distinct Mechanisms for Translational Repression 
Shintaro Iwasaki, Tomoko Kawamata, Yukihide Tomari 
Molecular Cell 
Volume 34, Issue 1, Pages (April 2009)
DOI: /j.molcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1
Both Ago1 and Ago2 Can Repress Translation of Their Target mRNAs
(A) Ago1-RISC or Ago2-RISC was programmed in embryo lysate for 30 min, and Renilla luciferase (RL) reporter mRNAs bearing zero, two, four, and eight let-7 target sites were translated in vitro for 1 hr together with firefly luciferase (FL) control mRNA. let-7/let-7∗ entered Ago1-RISC, whereas let-7, RL, and CXCR4 siRNAs entered Ago2-RISC (Figure S1A). The RL/FL luminescence was normalized to the value of no RISC programming. RL siRNA had a target site within the ORF of RL (positive control), while CXCR4 siRNA had no target site (negative control). The graph shows means and standard deviations from three independent trials.
(B) Stability of the reporter RL mRNAs bearing zero and eight target sites divided by that of the control FL mRNA normalized to the value of no RISC programming. Except for the RL siRNA, which cleaves a site within the ORF of RL, no significant change of mRNA levels was detected. The graph shows means and standard deviations quantified from three independent northern blot analyses, and representative data are shown in Figure S3A.
(C) Schematic representation of the RNase H assay used in (D).
(D) Ago1, but not Ago2, induces deadenylation of the target. Asterisks designate nonspecific signals. The positions of poly(A)+ and poly(A)− markers run on the same gel are indicated. Note that the length of the RNase H product differs depending on the number of the target sites.
Molecular Cell  , 58-67DOI: ( /j.molcel )
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3. Figure 2
Ago1 and Ago2 Employ Distinct Mechanisms for Translational Regulation
(A) Schematic representation of the RL target mRNAs used in (B) and (C).
(B) Ago1 inhibits translation primarily by deadenylation but can secondarily block a step after cap recognition.
(C) Ago2 represses translation by blocking the function of the cap structure. For (B) and (C), the RL/FL luminescence was normalized to the value of no RISC programming. The graph shows means and standard deviations from three independent trials. Black, m7G-capped RL mRNA bearing 8x target sites; red, A-capped reaper-IRES-containing RL mRNA bearing 8x target sites.
Molecular Cell  , 58-67DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3
Repression by Ago2 Is Independent of GW182, While Deadenylation by Ago1 Requires GW182 and ATP
(A) Translational repression of poly(A)+ target mRNA by Ago1 (solid circles), but not by Ago2 (open circles), was inhibited by the addition of GST-GW182(ΔC).
(B) Deadenylation by Ago1 requires GW182. GST-GW182(ΔC) blocked the Ago1-mediated deadenylation of the target mRNAs. GST or GST-GW182(ΔC) (12 μM) was present throughout the reaction.
(C) Deadenylation by Ago1 and GW182 requires ATP. Northern blot analysis after RNase H treatment is shown. CHX, 0.5 mM cycloheximide was added before the addition of target. −ATP, Ago1-RISC was programmed in the presence of ATP, and then ATP was depleted before the target was added.
Molecular Cell  , 58-67DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
Ago2-RISC Binds to eIF4E Only When Bound to a Cognate Target mRNA
(A) Ago1-RISC or Ago2-RISC was programmed in S2 lysate. m7G-capped RL mRNA bearing eight target sites (m7G-cap RL 8x) was added, and anti-eIF4E immunoprecipitation was then performed. Both Ago1 and Ago2 were coimmunoprecipitated with eIF4E, but Ago2 coimmunoprecipitation was significantly enhanced only when the cognate target for Ago2-RISC was present.
(B) Same as (A), but 100–800 mM KCl was supplemented in the washing buffer (which by itself contains 100 mM KOAc). Ago1 coimmunoprecipitation was eliminated, but the RNA-enhanced Ago2 coimmunoprecipitation was refractory to this treatment.
(C) RNase A treatment abolished Ago2 coimmunoprecipitation.
(D) Same as (A), but A-capped RL RNA bearing eight target sites (A-cap RL 8x) was used. A-cap RL 8x enhanced the Ago2 coimmunoprecipitation, to the same degree as m7G-cap RL 8x, indicating that the eIF4E-Ago2 interaction is not simply due to tethering by the target RNA.
(E) Same as (A), but m7G-capped or A-capped short RNA lacking the entire RL ORF and containing two target sites (m7G- or A-cap 2x) was used. The short RNAs, in which the region complementary to the let-7 seed sequence was mutated (mm), did not enhance the Ago2 coimmunoprecipitation.
Molecular Cell  , 58-67DOI: ( /j.molcel )
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6. Figure 5 Ago2-RISC Competes with eIF4G for eIF4E
(A) Trp-117 of eIF4E is essential for its association with Ago2. eif4e[RNAi] S2 lysate was supplemented with 200 nM GST-eIF4E(WT) or GST-eIF4E(W117A) and immunoprecipitated with anti-eIF4E antibody. Ago2 was efficiently coimmunoprecipitated only when GST-eIF4E(WT) was present.
(B) Ago2 binds to eIF4E independently of eIF4G or Cup. Addition of an increasing concentration of recombinant 4E-BP inhibited the coimmunoprecipitation of Ago2, Cup, and eIF4G by eIF4E antibody. The western blot signals were reasonably within a quantitative linear range (Figure S9).
(C) Quantification of (B). The data were fitted to the Hill equation. The Ago2-RISC-eIF4E interaction was more tolerant to 4E-BP than Cup-eIF4E and eIF4G-eIF4E interactions.
(D) Cap-radiolabeled target mRNA remained associated with eIF4E, but a significant amount dissociated from eIF4G when it was targeted by Ago2-RISC. In contrast, Ago1-RISC did not affect the association of the target with eIF4E and eIF4G. The amount of eIF4G-bound target divided by that of eIF4E-bound target was normalized to the value of CXCR4 siRNA, and means and standard deviations from four independent trials are shown by the graph in the inset. The p values were calculated using Student's two-tailed t test.
Molecular Cell  , 58-67DOI: ( /j.molcel )
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7. Figure 6
A Model for Distinct Translational Repression Mechanisms by Drosophila Ago Proteins
(A) Ago1-RISC represses translation primarily by ATP-dependent shortening of the poly(A) tail of its mRNA targets. Secondarily, Ago1-RISC can also block a step after cap recognition. These two processes are independent, but both require the P body component GW182.
(B) Ago2-RISC competitively inhibits translational initiation by blocking the interaction of eIF4E with eIF4G. This occurs only when Ago2-RISC binds to a cognate target mRNA.
Molecular Cell  , 58-67DOI: ( /j.molcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions