GW182 enhances let-7 miRNA-mediated translational repression. (A) Schematic representations of the firefly luciferase mRNAs FLuc-6xT and FLuc-6xTmut6. (B) Translation of capped and polyadenylated FLuc-6xT mRNA in the absence (−) or presence (+) of let-7. Cell extracts prepared from control 293F cells and 293F cells overexpressing Flag-Ago2 and Flag-GW182 (see Fig. 1A) were used in the combinations indicated above the figure. The FLuc and RLuc activities were measured, and the FLuc-to-RLuc activity ratio in the reaction without let-7 was set at 100. The data shown constitute an average of at least three independent experiments, with standard deviations. (C) Biotinylated FLuc-6xT mRNAs (capped and polyadenylated) were incubated with the mixed extract (Ago2 + GW182; see lane 1) in the absence (−) or presence (+) of let-7. The mRNAs were captured with SA-PMPs. (Left panel) The proteins bound to the mRNAs were detected using an anti-Flag M2 antibody (lane 1, Ago2 + GW182 extract; lanes 2–4, pull-down). The sizes (in kilodaltons) of the molecular markers (M) are indicated on the left. (Right panel) The mRNAs captured with SA-PMPs were detected by dot hybridization (see the text).

Let-7 miRNAs direct deadenylation of the target mRNAs. (A) Analyses of capped and polyadenylated FLuc-6xT (6xT) and FLuc-6xTmut6 (mut6) mRNAs by Northern blotting. As molecular markers, nonadenylated and polyadenylated transcripts (capped) were loaded in lanes 1 and 2, respectively. After 60 min of translation in the absence (−) or presence (+) of let-7 and anti-let-7, the RNAs were extracted from the reaction mixture, and similar amounts were loaded in each lane, from lanes 3–6. In the middle panel, the extracted RNAs, as in the top panel (6xT), were annealed with oligo(dT) and subjected to RNaseH digestion. (B) Polyadenylated ApppG-FLuc-6xT and ApppG-capped EMCV IRES-FLuc-6xT mRNAs were translated in the absence (−) or presence (+) of let-7. In lane M, polyadenylated (longer) and nonadenylated (shorter) transcripts were loaded as markers. (C) PAT assay of polyadenylated FLuc-6xT mRNAs extracted after 60 min of translation in the absence (−) or presence (+) of let-7. The structures of the 5′ termini are indicated above the figures. (D) Capped and polyadenylated FLuc-6xT mRNAs were incubated in the translation reaction mixture for 60 min in the presence or absence of chx. Then, RNAs were extracted and FLuc-6xT mRNAs were detected by Northern blotting. The minus (−) and plus (+) signs above the lanes indicate the absence and presence of let-7. In lane M, polyadenylated (longer) and nonadenylated (shorter) transcripts were loaded as markers.

Overexpression of Ago2 stimulates the processing of let-7 pre-miRNA. (A) Expression of Dicer, TRBP2, Ago2, and GW182 in HEK293F cells. The expressed proteins were detected by Western blotting using an anti-Flag M2 antibody (Sigma). The sizes (in kilodaltons) of the molecular markers (see lane M) are indicated on the left. (B) Processing assay of let-7 pre-miRNA. Chemically synthesized let-7 pre-miRNA was incubated with extracts prepared from 293F cells, or the mixed extracts composed of the extracts prepared from 293F cells overexpressing Dicer, TRBP2, Ago2, or GW182, at the ratios indicated above the figure. The RNA was then extracted, and let-7 was detected by Northern blotting. Markers are a 60-nt (60nt) let-7 pre-miRNA and a 21-nt (21nt) let-7 siRNA. Five-hundred nanograms of RNA were loaded in each lane, except for the markers.

The cap and poly(A) tail are important for the let-7-mediated translational repression. In the following experiments, Ago2 + GW182 extracts (see the text) were used. The FLuc and RLuc activities were measured, and the FLuc-to-RLuc activity ratio in the reaction without let-7 was set at 100, except in the experiment shown in D. The data shown constitute an average of at least three independent experiments, with standard deviations. (A) Capped FLuc-6xT and FLuc-6xTmut6 mRNAs with or without a poly(A) tail were translated in the absence (−) or presence (+) of let-7. (B) Capped and polyadenylated FLuc-6xT mRNA was translated in the absence (−) or presence (+) of let-7 and anti-let-7 (Ambion). (C) ApppG-FLuc-6xT and ApppG-capped EMC VIRES-FLuc-6xT mRNAs with or without a poly(A) tail were translated in the absence (−) or presence (+) of let-7. (D) Comparison of translation efficiencies of different constructs of FLuc-6xT reporter mRNAs in the absence of let-7. The average number of light units for the Cap/poly(A) mRNA was ∼7,000,000.

Strong correlation between mRNA deadenylation and translational repression. (A) Time-course analysis of luciferase synthesis. Capped and polyadenylated FLuc-6xT mRNA and capped RLuc mRNA were translated. Aliquots were taken for luciferase assays at time points 0, 20, 30, 40, and 60 min from the start of translation. (B) In parallel with the analyses of luciferase activities, RNAs were extracted from the aliquots taken at each time point, and were subjected to Northern hybridization analyses using the FLuc probe.