MicroRNAs Mediate Gene Silencing via Multiple Different Pathways in Drosophila  Takashi Fukaya, Yukihide Tomari  Molecular Cell  Volume 48, Issue 6, Pages 825-836 (December 2012) DOI: 10.1016/j.molcel.2012.09.024 Copyright © 2012 Elsevier Inc. Terms and Conditions

Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Ago1-RISC Represses Translation Independently of GW182 In Vivo (A) Schematic representation of Rluc-let-7-poly(A) and Rluc-let-7-A114-N40-HhR reporter constructs used in (B)–(E). (B) Luciferase reporter assay for Rluc-let-7-poly(A) and Rluc-let-7-A114-N40-HhR in the absence of GW182 or Ago1. RNAi against GFP was used as a mock. The Rluc/Fluc luminescence was normalized to the value of no let-7 expression. The mean values ± SD from three independent experiments are shown (similarly hereinafter). Silencing was abolished in ago1[RNAi], but not in gw182[RNAi]. See (D) for the procedure for knockdown and the reporter assay. (C) Northern blot analysis of representative RNA samples isolated from S2 cells shown in (B). Knockdown of GW182 or Ago1 inhibited miRNA-mediated deadenylation/decay of Rluc-let-7-poly(A), while Rluc-let-7-A114-N40-HhR was unaffected. (D) S2 cells were soaked in dsRNAs corresponding to the protein coding sequence of gw182 or ago1 on day 1, the Rluc and Fluc reporter plasmids together with let-7-expressing plasmid or empty plasmid were transfected on day 3, and the luciferase assay was performed on day 6. Ago1 or GW182 was efficiently depleted, from transfection through the luciferase assay. (E) Kinetics of translational repression of Rluc-let-7-A114-N40-HhR in mock (gfp[RNAi]) or gw182[RNAi] S2 cells. GW182 slightly augments translational repression by Ago1-RISC in early time points. See also Figure S1. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Ago1-RISC Represses Translation Independently of GW182 In Vitro (A) Schematic representation of mini-let-7-A114 and Rluc-let-7-A114-N40 reporter constructs used in (B)–(F). (B) Luciferase assay for Rluc-let-7-A114-N40 in gw182[RNAi] lysate. RNAi against GFP was used as a mock. The Rluc/Fluc luminescence was normalized to the value of no Ago1-RISC programming. GW182 was dispensable for pure translational repression by Ago1-RISC. (C) Deadenylation assay for mini-let-7-A114 in gw182[RNAi] lysate. Deadenylation by Ago1-RISC was abolished in the absence of GW182. (D) Western blotting analysis for the samples used in (B) and (C). (E) Pure translational repression of Rluc-let-7-A114-N40 in naive lysate and Ago1-depleted lysate. Translational repression was critically affected by the amount of free Ago1 available for exogenous RISC programming. (F) Deadenylation of mini-let-7-A114 in naive and Ago1-depleted lysates. Deadenylation was limited by the amount of free Ago1. (G) Western blotting analysis for the samples used in (E) and (F). See also Figure S2. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Neither GW182 nor CCR4-NOT Complex Is Required for Translational Repression by Ago1-RISC (A) CAF1, NOT1, and/or GW182 were knocked down by RNAi. FLAG-Ago1 was expressed in all samples. RNAi against GFP was used as a mock. (B) Deadenylation of mini-let-7-A114 in the series of knockdown lysates in (A). GW182, CAF1, and NOT1 are indispensable for Ago1-RISC-mediated deadenylation. (C) Translational repression of Rluc-let-7-A114-N40 in the series of knockdown lysates in (A). GW182, CAF1, and NOT1 are dispensable for Ago1-RISC-mediated translational repression. See also Figure S3. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Ago1-RISC Blocks Early Translation Initiation Independently of GW182 (A) Schematic representation of deadenylation-resistant sORF-let-7-A114-N40 reporter construct used for sucrose density gradient analysis. (B) UV profile for detection of 48S initiation complex (left), 80S initiation complex (middle), or polysomes and pseudopolysomes (right). Gradient conditions are shown at the bottom. Peaks for 40S, 60S, and 80S ribosomal complexes and polysomes are indicated. (C) Sucrose density gradient analysis with sORF-let-7-A114-N40 in the presence (red) or absence (black) of let-7-programmed Ago1-RISC. Formation of 48S (left) and 80S (middle) initiation complexes was inhibited by Ago1-RISC. Targeting of Ago1-RISC induced formation of dense pseudopolysomes (right). (D) Sucrose density gradient analysis with sORF-let-7-A114-N40 in gw182[RNAi] lysate in the presence (red) or absence (black) of let-7-programmed Ago1-RISC. Formation of 48S (left) and 80S (middle) initiation complexes was inhibited by Ago1-RISC even in the absence of GW182. Pseudopolysomes were collapsed in gw182[RNAi] lysate. See also Figure S4. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 eIF4A-Independent Noncanonical Translation Is Immune to Translational Repression by Ago1-RISC (A) Schematic representation of Acap-Loop-Rluc-BoxB-let-7-A114-N40 reporter construct used in (C) and (D). (B) Expression of λN-LacZ or λN-eIF4G together with FLAG-Ago1 in S2 cells. (C) Luciferase reporter assay for Acap-Loop-Rluc-BoxB-let-7-A114-N40 in λN-eIF4G expressing lysate. The Rluc/Fluc luminescence was normalized to the value of λN-LacZ tethering. λN-eIF4G tethering activated translation by ∼10-fold. (D) Translational repression of Acap-Loop-Rluc-BoxB-let-7-A114-N40 in λN-eIF4G expressing lysate. Ago1-RISC blocks a step downstream of eIF4G. (E) Schematic representation of Acap-reaper-Rluc-let-7-A114-N40 reporter construct used in (F). (F) Translational repression of Acap-reaper-Rluc-let-7-A114-N40 in the presence of hippuristanol. Ago1-RISC cannot repress eIF4A-independent translation. See also Figure S5. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 6 Direct Tethering of GW182 Induces Deadenylation and Translational Repression (A) Schematic representation of mini-BoxB-A114 and Rluc-BoxB-A114-N40 reporter constructs used in (B)–(G). (B) Deadenylation of mini-BoxB-A114 by tethered GW182 with or without Ago1 immunodepletion. LacZ tethering was used as a negative control. Tethered GW182 mediated deadenylation independently of Ago1. (C) Pure translational repression of Rluc-BoxB-A114-N40 by tethered GW182. LacZ tethering was used as a negative control. Tethered GW182 induced translational repression independently of Ago1. (D) λN-GW182 or λN-LacZ was expressed in S2 cells, and Ago1 was immunodepleted from the lysate by immobilized anti-Ago1 antibody or by beads alone as a mock. (E) Deadenylation of mini-BoxB-A114 by tethered GW182 in the presence or absence of CAF1. LacZ tethering was used as a negative control. CAF1 was required for deadenylation by tethered GW182. (F) Translational repression of Rluc-BoxB-A114-N40 by tethered GW182. LacZ tethering was used as a negative control. Tethered GW182 induced translational repression independently of CAF1. (G) λN-GW182 or λN-LacZ was expressed in S2 cells and CAF1 was knocked down by RNAi. RNAi against GFP was used as a mock. CAF1 knockdown did not affect the expression level of λN-GW182. See also Figure S6. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 7 GW182-Mediated Translational Repression Blocks Early Translation Initiation (A) Schematic representation of deadenylation-resistant sORF-A114-N40 and sORF-BoxB-A114-N40 reporter constructs used in (B). (B) Sucrose density gradient analysis with sORF-BoxB-A114-N40 tethered with λN-GW182 (red) and sORF-A114-N40 (black). GW182 tethering inhibited formation of 48S (left) and 80S (middle) initiation complexes, while inducing formation of pseudopolysome formation (right). (C) Multiple silencing pathways employed by miRNAs. Ago1-RISC can repress early translation initiation independently of GW182 (1). GW182, recruited by Ago1-RISC to target mRNAs, mediates deadenylation via the CCR4-CAF1-NOT deadenylase complex (2), pure repression of early translation initiation (3), and formation of pseudopolysomes (4). Contribution of these multiple pathways to the overall outcome should vary depending on biological systems. See also Figure S7. Molecular Cell 2012 48, 825-836DOI: (10.1016/j.molcel.2012.09.024) Copyright © 2012 Elsevier Inc. Terms and Conditions