Immunopurified myc-Ago2, both wild type and catalytically inactive, process pre-miR-30a and artificial PML RNA. (A, left panel) Primary and secondary structure of pre-miR-30a used in the processing reactions. The 5′-end-labeled mature miR-30a-5p generated after processing is shown. Red asterisk indicates the radiolabeled 5′-phosphate. (Right panel) Immunoprecipitations were performed with anti-myc antibody from 293T cells transfected with myc-tagged wild-type Ago2 (myc-Ago2-WT), with a catalytically inactive Ago2 mutant (myc-Ago2-D669A), or with myc-hnRNPQ3 (negative control). The immunoprecipitates were incubated with 5′-end-radiolabeled synthetic pre-miR-30a. Reaction products were analyzed on a 15% UREA-PAGE and visualized by autoradiography. (B, left panel) Primary and secondary structure of an artificial PML RNA (PML-GL3) used in the processing reactions. (Right panel) Processing reactions with 5′-end-radiolabeled PML-GL3.

Asymmetric loading of pre-miRNA processing products on RISC. (A) pre-let-7a-3 processing assay. 5′-end-labeled pre-let7a-3 (lane 1) or 3′-end-labeled pre-let-7a-3 (lane 3) was incubated with myc-Ago2 beads, and the reaction products were analyzed on a 15% UREA-PAGE; the miRNAs produced (lanes 2,4) are indicated. (B) Sequence of pre-let-7a-3, its processing products (let-7a and let-7a^⋆), and the corresponding RNA targets (let-7a-Target and let-7a^⋆-Target). The radiolabeled 5′-phosphate of let-7a^⋆-Target and pCp (3′-end) of let-7a-Target are shown in red. (C) RISC loading assays with pre-let7a-3. (Top panel) 3′-end-labeled let-7a-Target (unreacted in lane 1) was incubated with mock preincubated myc-Ago2 beads (lane 2) and with myc-Ago2 beads that were preincubated with pre-let7a-3 (lane 3). (Bottom panel) 5′-end-labeled let-7a^⋆-Target (unreacted in lane 1) was incubated with mock myc-Ago2 beads (lane 2) and with myc-Ago2 beads that were preincubated with pre-let7a-3 (lane 3).

ATP does not affect the rate of target cleavage. (Top) RISCs were assembled with myc-Ago2 immunoprecipitates and cold PML-GL3 (as described in Fig. 3) and were incubated with 5′-end-labeled GL3-target RNA (10 nM) in the absence or presence of 1 mM ATP/0.2 mM GTP. (Bottom) Samples were taken at indicated times, and product formation was quantitated with PhosphorImager and plotted against reaction time. The fitting of rates was done with Kaleidagraph software.

Working model of human RISC assembly from pre-miRNAs.

Immunopurified Flag-Ago2 contains Dicer and displays pre-miRNA processing activity. (A) 293 cells were stably transfected with Flag-Ago2 under the control of a tetracycline-inducible promoter. Immunoprecipitations (IP) were performed with anti-Flag antibody from noninduced (-) and from tetracycline-induced (+) 293 cells, and the immunoprecipitates were resolved on a 4%-12% NuPAGE and stained with silver. (B) Immunoprecipitates from A were analyzed by Western blot with the indicated antibodies. (C) Immunoprecipitates from A were incubated with a synthetic pre-miR-30a (sequence shown in Fig. 2A) containing a radiolabeled 5′-phosphate, and the reaction products were analyzed on a 15% UREA-PAGE and visualized by autoradiography.

Assembly of functional RISCs from immunopurified myc-Ago2 and synthetic pre-miRNA. (A) Schematic of the two-step experimental protocol used to test RISC loading by pre-miRNA. (B) RISC loading assay performed with cold, 5′-phosphorylated pre-miR-30a. (B, top panel) Sequences of the two RNAs used (pre-miR-30a and cognate Target for miR-30a) and of the mature miRNA (mir-30a-5p) produced from processing of pre-miR-30a. Red asterisk indicates the radiolabeled 5′-phosphate of the RNA target; the lightning bolt shows the position of mir-30a-5p-mediated target RNA cleavage. (Bottom panel) Agarose beads containing immunopurified myc-Ago2, either wild-type (WT) or the catalytically inactive mutant D669A (669), were first incubated in the presence of unlabeled pre-miR-30a (lanes 4,5) or in its absence (lanes 2,3). The beads were then washed and incubated with 5′-end-labeled miR-30a-Target. The first lane (-) shows input target RNA. (C) RISC loading assay performed with cold, 5′-phosphorylated PML-GL3. (Top panel) Sequences of the RNAs used and of the mature GL3-24 produced from processing of PML-GL3. (Bottom panel) Agarose beads containing myc-Ago2, wild type (WT) or D669A mutant (669), were first incubated with unlabeled PML-GL3. The beads were then washed and incubated either with 5′-end-labeled GL3-Target (lanes 2,3) or with 5′-end-labeled miR-30a-Target (lane 5), which served as the target recognition-specificity control. Lanes 1 and 4 (-), show input target RNAs. RNA was analyzed by 17.5% UREA-PAGE and visualized by autoradiography.

Immunopurified myc-Ago2 complexes can generate RISC from pre-miRNA, but not from corresponding siRNA duplex, regardless of ATP amounts. (A, left panel) Synthetic RNAs used for RISC assembly by immunopurified myc-Ago2-WT; all RNAs contained 5′-phosphates. Sequences shown in green are complementary to the GL3-Target RNA. (Right panel) Agarose beads containing immunopurified, wild-type myc-Ago2 were incubated with cold, PML-GL3 RNA in the absence (lane 2) or presence of 1 mM ATP and 0.2 mM GTP (lane 3); or with GL3 siRNA duplex without (lane 4) or with (lane 5) ATP/GTP; or with single-stranded GL3-24 RNA, without (lane 6) or with (lane 7) ATP/GTP. The beads were then washed and incubated with 5′-end-radiolabeled GL3-Target RNA. The first lane shows unreacted GL3-Target RNA. RNA was analyzed as in A. (B) Agarose beads containing immunopurified, wild-type myc-Ago2 complexes were depleted of ATP by extensive washings. The beads were used in RISC loading and cognate target cleavage reactions with cold, 5′-phosphorylated and gel-purified pre-miR030a or PML-GL3 in the absence (lanes 2,6) or the presence (lanes 3,7) of 2 mM ATP and 0.4 mM GTP. The 5′-end-labeled RNA targets (lanes 1,5 show unreacted targets) were also gel-purified. A synthetic 5′-end-labeled 18-nt RNA oligo in lane 4 served as a size marker. RNA was analyzed by 17.5% UREA-PAGE and visualized by autoradiography.

RISC activity dissociates from Dicer. 293T cells were doubly transfected with plasmids that express V5-Dicer and myc-Ago2. Immunoprecipitations were done with anti-V5 or with anti-myc antibodies. The immunoprecipitates were used in a modified RISC loading assay in which, after the pre-miRNA processing reaction, the beads and the supernatants were first separated and then tested for cognate target RNA cleavage. (A, left panel) Schematic of the assay. (Right panel) V5-Dicer immunoprecipitates were used in the modified RISC loading assay with cold, 5′-phosphorylated pre-miR-30a. The 5′-end-labeled mir-30a-Target (unreacted in lane 1) was incubated with the beads (lane 2) or with the supernatant (lane 3) after the processing reactions, as well as with a mock supernatant, from a V5-Dicer immunoprecipitate where no substrate had been added to the processing step, but where pre-miR30a was added to the supernatant prior to the target cleavage reaction (lane 4). (B) Cold, 5′-phosphorylated PML-GL3 was used as a processing substrate with the V5-Dicer immunoprecipitates in the modified RISC loading assay. The 5′-end-labeled PML-GL3-Target (unreacted in lane 1) was incubated with the beads (lane 2) or with the supernatant (lane 3) from the processing reactions, as well as with a mock supernatant, from a V5-Dicer immunoprecipitate where no substrate had been added to the processing step, but where prior to the target cleavage reaction, cold, 5′-phosphorylated PML-GL3 (PML; lane 4), 5′-phosphorylated GL3 siRNA duplex (si; lane 5), or 5′-phosphorylated GL3-24 (as; lane 6) was added. (C, left panel) Schematic of the assay. (Right panel) The myc-Ago2 immunoprecipitates were used in the modified RISC loading protocol, with cold, 5′-phosphorylated PML-GL3 as a processing substrate. The 5′-end-labeled PML-GL3-Target was incubated with the beads (lane 1) or with the supernatant (lane 2) from the processing reactions.