1. MicroRNAs Block Assembly of eIF4F Translation Initiation Complex in Drosophila 
Takashi Fukaya, Hiro-oki Iwakawa, Yukihide Tomari 
Molecular Cell 
Volume 56, Issue 1, Pages (October 2014)
DOI: /j.molcel
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2. Molecular Cell 2014 56, 67-78DOI: (10.1016/j.molcel.2014.09.004)
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3. Figure 1
Depletion of eIF4A Attenuated miRNA-Mediated Translational Repression in S2 cells
(A) Schematic representation of the Renilla luciferase (Rluc) reporter mRNA with let-7 binding sites (Rluc-let-7-A114-N40-HhR) transcribed from the pAWH-Rluc-let-7-A114-N40-HhR plasmid. Rluc-let-7-A114-N40-HhR harbors a 114-nt poly(A) sequence (A114) internalized by a 40-nt unrelated sequence (N40) in the 3′ UTR followed by a hammer head ribozyme (HhR) that generates the 3′ end by self-cleavage.
(B) S2 cells were soaked in dsRNAs corresponding to the protein-coding region of eIF4E, eIF4G, or eIF4A on day 1, then transfected with the Rluc and Fluc reporter plasmids together with let-7-expressing plasmid or empty plasmid on day 3. The luciferase assay was performed on day 6. Each eIF4F components was efficiently depleted during day 3 to day 6. Mock indicates naive S2 cells.
(C) Luciferase reporter assay for Rluc-let-7-A114-N40-HhR in the S2 cells with each of the eIF4F components knocked down. Depletion of eIF4A, but not that of eIF4E or eIF4G, attenuated miRNA-mediated translational repression. The Rluc/Fluc luminescence was normalized to the value of no let-7 expression (−let-7). The mean values ± SD from three independent experiments are shown.
(D) Basal translation in S2 cell was severely impaired by depletion of eIF4F components. The Rluc luminescence (without being divided by the Fluc luminescence) was normalized to the value of the control dsRNA-treated cells (gfp[RNAi]). The mean values ± SD from three independent experiments are shown.
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4. Figure 2 Detection of eIF4E and eIF4A by UV Crosslinking
(A) Schematic representation of sORF-A114-N40 reporter construct used in (C)–(F).
(B) 5′ UTR sequences of the reporter constructs. Positions of the 4-thio-U (4SU) and the RNaseA digestion site closest to the 5′ cap structure are indicated in red and blue, respectively.
(C) Detection of eIF4E by UV crosslinking. Cap-radiolabeled sORF-2U-A114-N40 containing 4-thio-U was incubated in S2 cell lysate, crosslinked by UV light, immunoprecipitated by anti-eIF4E antibody, and then analyzed by SDS-PAGE. eIF4E was efficiently crosslinked to 4-thio-U at 2 nt from the cap.
(D) Detection of eIF4A by UV crosslinking. Cap-radiolabeled sORF-13U-A114-N40 containing 4-thio-U was incubated in S2 cell lysate, crosslinked by UV light, immunoprecipitated with anti-eIF4A antibody, and then analyzed by SDS-PAGE. eIF4A was efficiently crosslinked to 4-thio-U at 13 nt from the cap.
(E) Crosslinking of eIF4E was blocked by cap competitor m7GpppG, but not by cap analog ApppG or eIF4A inhibitor hippuristanol.
(F) Crosslinking of eIF4A was blocked by eIF4A inhibitor hippuristanol or cap competitor m7GpppG, but not cap analog ApppG.
(G) Schematic representation of sORF-A0 reporter construct used in (H) and (I).
(H) UV crosslinking of eIF4E with or without the poly(A) tail. Loss of the poly(A) tail decreased crosslinking of eIF4E.
(I) UV crosslinking of eIF4A with or without the poly(A) tail. Loss of the poly(A) tail decreased crosslinking of eIF4A.
See also Figure S1.
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5. Figure 3 Ago1-RISC Induces Dissociation of eIF4A
(A) Schematic representation of sORF-let-7-A114-N40 and Rluc-let-7-A114-N40 reporter constructs used in (B)–(E). The 5′ UTR sequences of the 2U and 13U reporter constructs are shown in Figure 2B.
(B) Luciferase reporter assay for Rluc-2U or 13U-A114-N40 (−target site) and Rluc-2U or 13U-let-7-A114-N40 (+target site). The Rluc/Fluc luminescence was normalized to the value of no RISC programming. The mean values ± SD from three independent experiments are shown. Ago1-RISC specifically induced translational repression of reporter mRNA containing target sites for let-7 miRNA.
(C) UV crosslinking of eIF4E under Ago1-RISC-mediated translational repression. sORF-2U-A114-N40 (−target site) or sORF-2U-let-7-A114-N40 (+target site) was incubated in S2 cell lysate in the presence or absence of let-7-programmed Ago1-RISC and crosslinked with UV light. Ago1-RISC did not affect crosslinking of eIF4E.
(D) UV crosslinking of eIF4A under Ago1-RISC-mediated translational repression. sORF-13U-A114-N40 (−target site) or sORF-13U-let-7-A114-N40 (+target site) was incubated in S2 cell lysate in the presence or absence of let-7-programmed Ago1-RISC and crosslinked with UV light. Ago1-RISC blocked crosslinking of eIF4A.
(E) Change in the amount of crosslinked eIF4E and eIF4A by let-7-programmed Ago1-RISC relative to no RISC programming. The signal intensity of the bands in (C) and (D) was quantified, and the ratio between the values in the presence or absence of let-7-programmed Ago1-RISC was calculated. The mean values ± SD from three independent experiments are shown.
(F) Schematic representation of reporter mRNAs with Renilla luciferase ORF (Rluc-vha68-1-A114-N40) or short ORF (sORF-vha68-1-A114-N40) bearing the 3′ UTR of natural vha68-1 mRNA containing two miR-9b binding sites used in (G)–(J). The 5′ UTR sequences of the 2U and 13U reporter constructs are shown in Figure 2B.
(G) Luciferase reporter assay for Rluc-2U or 13U-vha68-1-A114-N40. Ago1-RISC induced translational repression of reporter RNAs with natural miRNA-target sites in the presence of miR-9b. The mean values ± SD from three independent experiments are shown.
(H) UV crosslinking of eIF4E for sORF-2U-vha68-1-A114-N40 in the presence or absence of miR-9b-programmed Ago1-RISC. Ago1-RISC did not affect crosslinking eIF4E.
(I) UV crosslinking of eIF4A for sORF-13U-vha68-1-A114-N40 in the presence or absence of miR-9b-programmed Ago1-RISC. Ago1-RISC induced dissociation of eIF4A.
(J) Change in the amount of crosslinked eIF4E and eIF4A by miR-9b-programmed Ago1-RISC relative to no RISC programming in (H) and (I). The mean values ± SD from three independent experiments are shown.
See also Figures S2, S4, and S5.
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6. Figure 4
Ago1-RISC Induces Dissociation of eIF4A after Assembly of eIF4F Complex on the Target mRNAs
(A) Scheme for the experimental procedure. Target mRNAs and let-7/let-7∗ duplexes were separately incubated in the FLAG-Ago1 S2 lysate for eIF4F assembly and for RISC programming, respectively. These lysates were then mixed together and exposed to UV irradiation immediately or after 30 min incubation.
(B) UV crosslinking of eIF4A. Ago1-RISC induced dissociation of eIF4A from preassembled eIF4F complex on the target mRNA.
(C) Change in the amount of crosslinked eIF4A by let-7-programmed Ago1-RISC relative to no RISC programming in (B). The mean values ± SD from three independent experiments are shown.
See also Figures S4 and S5.
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7. Figure 5
Addition of Excess eIF4A Attenuated eIF4A Dissociation by Ago1-RISC
(A) Western blotting for exogenous eIF4E added in excess.
(B) Luciferase assay for Rluc-13U-let-7-A114-N40 in the presence of 1 μM, 5 μM, or 10 μM His-eIF4E. Addition of excess eIF4E did not affect the efficiency of translational repression by Ago1-RISC. The mean values ± SD from three independent experiments are shown.
(C) Western blotting for exogenous eIF4A added in excess.
(D) Luciferase assay for Rluc-13U-let-7-A114-N40 in the presence of 1 μM, 5 μM, or 10 μM His-eIF4A. Addition of excess eIF4A partially relieved translational repression by Ago1-RISC. The mean values ± SD from three independent experiments are shown.
(E) UV crosslinking of eIF4E in the presence of 1 μM, 5 μM, or 10 μM His-eIF4E. Addition of excess eIF4E increased eIF4E crosslinking.
(F) Change in the amount of crosslinked eIF4E by let-7-programmed Ago1-RISC relative to no RISC programming in (E). The mean values ± SD from three independent experiments are shown.
(G) UV crosslinking of eIF4A in the presence of 1 μM, 5 μM, or 10 μM His-eIF4E. Addition of excess eIF4E did not affect eIF4A dissociation by Ago1-RISC.
(H) Change in the amount of crosslinked eIF4A by let-7-programmed Ago1-RISC relative to no RISC programming in (G). The mean values ± SD from three independent experiments are shown.
(I) UV crosslinking of eIF4E in the presence of 1 μM, 5 μM, or 10 μM His-eIF4A. Addition of excess eIF4A did not affect eIF4E crosslinking.
(J) Change in the amount of crosslinked eIF4E by let-7-programmed Ago1-RISC relative to no RISC programming in (I). The mean values ± SD from three independent experiments are shown.
(K) UV crosslinking of eIF4A in the presence of 1 μM, 5 μM, or 10 μM His-eIF4A. Addition of excess eIF4A attenuated the reduction of eIF4A crosslinking by Ago1-RISC.
(L) Change in the amount of crosslinked eIF4A by let-7-programmed Ago1-RISC relative to no RISC programming in (K). The mean values ± SD from three independent experiments are shown.
See also Figures S3–S5.
Molecular Cell  , 67-78DOI: ( /j.molcel )
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8. Figure 6
Direct Tethering of GW182 Induces Dissociation of eIF4E and eIF4A
(A) Schematic representation of sORF-BoxB-A114-N40 and Rluc-BoxB-A114-N40 reporter constructs used in (C)–(F). The 5′ UTR sequences of the 2U and 13U reporter constructs are shown in Figure 2B.
(B) Western blotting analysis for the S2 cell lysate used in (C)–(K).
(C) Pure translational repression of Rluc-2U or 13U-BoxB-A114-N40 by tethered GW182. The Rluc/Fluc luminescence was normalized to the value of LacZ tethering. Tethered GW182 induced translational repression. The mean values ± SD from three independent experiments are shown.
(D) UV crosslinking of eIF4E under translational repression by tethered GW182. sORF-2U-A114-N40 (−BoxB site) or sORF-2U-BoxB-A114-N40 (+BoxB site) was used for crosslinking. LacZ tethering was used as a negative control. Tethered GW182 blocked crosslinking of eIF4E.
(E) UV crosslinking of eIF4A under translational repression by tethered GW182. sORF-13U-A114-N40 (−BoxB site) or sORF-13U-BoxB-A114-N40 (+BoxB site) was used. Tethered GW182 blocks crosslinking of eIF4A.
(F) Change in the amount of crosslinked eIF4E and eIF4A by direct tethering of GW182 or LacZ relative to mock tethering. The signal intensity of the bands in (D) and (E) was quantified, and the ratio between the values in the presence or absence of the BoxB binding site was calculated. The mean values ± SD from three independent experiments are shown.
(G) Schematic representation of sORF-BoxB-A0 and Rluc-BoxB-A0 reporter constructs used in (H)–(K). The 5′ UTR sequences of the 2U and 13U reporter constructs are shown in Figure 2B.
(H) Translational repression of Rluc-2U or 13U-BoxB-A0 by tethered GW182. Tethered GW182 induced translational repression of the poly(A)− mRNA. The mean values ± SD from three independent experiments are shown.
(I) UV crosslinking of eIF4E for the poly(A)− mRNA under repression by tethered GW182. sORF-2U-A0 (−BoxB site) or sORF-2U-BoxB-A0 (+BoxB site) was used. Tethered GW182 blocked crosslinking of eIF4E even without the poly(A) tail.
(J) UV crosslinking of eIF4A for the poly(A)− mRNA under repression by tethered GW182. sORF-13U-A0 (−BoxB site) or sORF-13U-BoxB-A0 (+BoxB site) was used. Tethered GW182 blocked crosslinking of eIF4A even without the poly(A) tail.
(K) Change in the amount of crosslinked eIF4E and eIF4A by direct tethering of GW182 or LacZ relative to mock tethering. The signal intensity of the bands in (I) and (J) was quantified, and the ratio between the values in the presence or absence of the BoxB binding site was calculated. The mean values ± SD from three independent experiments are shown.
See also Figure S6.
Molecular Cell  , 67-78DOI: ( /j.molcel )
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9. Figure 7 miRNAs Mediate Dissociation of eIF4A but Not eIF4E
(A) Luciferase reporter assay for Rluc-2U-let-7-A114-N40 and Rluc-13U-let-7-A114-N40 in the presence of antisense 2′-O-methylated oligonucleotides (ASO) to let-7 (anti-let-7) or miR-9b (anti-miR-9b) in the let-7 expression lysate. Translation of reporter RNAs was derepressed by the addition of anti-let-7. The Rluc/Fluc luminescence was normalized to the value in the presence of anti-miR-9b. The mean values ± SD from three independent experiments are shown.
(B) UV crosslinking of eIF4E in the presence of ASOs. Addition of ASOs did not affect eIF4E crosslinking.
(C) UV crosslinking of eIF4A in the presence ASOs. Addition of anti-let-7 increased eIF4A crosslinking.
(D) Quantification of eIF4E and eIF4A crosslinking in the presence of ASOs. The signal intensity was normalized to the value in the presence of anti-miR-9b. The mean values ± SD from three independent experiments are shown.
See also Figure S7.
Molecular Cell  , 67-78DOI: ( /j.molcel )
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