RNA-binding and catalytic activity of the reconstituted RLC. (A) Nitrocellulose filter-binding assay: ≈1 nM radiolabeled Dmpre-let7 hairpin RNA was incubated with indicated concentrations of RLC, yielding a measured K_d of 15 nM. (B) Dicing assay: 200 nM RLC or 200 nM human Dicer (as a positive control) was incubated with ≈10 nM 5′ end ³²P-labeled Dmpre-let7 RNA. (C) Slicing assay: 200 nM RLC or 200 nM human Ago2 (as a positive control) was incubated with 250 nM guide RNA and then was added to ≈10 nM 5′ end ³²P-labeled 37-nt A RNA annealed with 37-nt B. Slicing product, 25 nt; dicing product, 16 nt.

RNA-dependent disassembly of the human RLC complex. (A) Coomassie-stained SDS/PAGE gel showing reconstitution of the RLC complex by using FLAG-tagged Argonaute 2 (FLAG-hAgo2) or FLAG-tagged Dicer (FLAG-hDcr). Samples were assembled by incubation of RLC components as described in Fig. 1 and were isolated by using Agarose bead-coupled anti-FLAG antibodies. RLC components (hDcr, hAgo2, and TRBP) and antibodies (L-Ab and S-Ab) are labeled. (B) Phosphorimage of a denaturing polyacrylamide gel showing slicing activity of bead-bound (beads) and supernatant (spn) samples from A after incubation with 5′ end ³²P-labeled 37-nt A target (substrate) RNA and unlabeled Dmpre-let hairpin RNA. Target and Dmpre-let RNAs incubated in buffer alone (mock) provided controls for nonspecific degradation. Mobilities of RNA substrate, 25-nt slicing product, and a 21-nt RNA marker oligo are indicated on the right. (C) Coomassie-stained SDS/PAGE gel analysis of FLAG-hDcr/hAgo2/TRBP samples prepared as in A after incubation in buffer alone (−) or buffer containing duplex RNA substrate 37-nt A and B (+). The buffer contains no Mg²⁺ but is supplemented with 5 mM EDTA.

In vitro assembly of the human RLC. (A) Superose 6 size-exclusion chromatography elution profiles are shown for individual samples of human Dicer, Ago2, and TRBP or a preincubated mixture of the three proteins containing an ≈2-fold molar excess of Ago2 and TRBP. The chromatogram is labeled to indicate RLC (red asterisk), fractions of the RLC peak (red line), and fractions containing unincorporated Ago2 and TRBP (green line). (B) SDS/PAGE gel analysis of fractions from A and labeled as in A. In some preparations, we observed a contaminating band at ≈70 kDa that was identified as insect Hsp70-4 by MS. (C) Electrospray MS of the intact RLC complex. (Lower) Mass spectrum of the intact heterotrimeric RLC complex (purple star) confirms the presence of Dicer, Ago2, and TRBP subunits with unit stoichiometry. The charge-state series allows the molecular mass to be measured as 371.129 ± 95 Da, indicating the presence of an additional component with a molecular mass 11 kDa higher than expected. (Inset) This larger mass is likely due to an RNA bound to Ago 2, because the Ago 2 alone has a molecular mass of 109.950 ± 53 Da. At m/z ≈7,500, the Dicer:TRBP (orange circle) heterodimer also was observed (molecular mass of 259.212 ± 62 Da), indicating its formation in solution. (Upper) MS/MS analysis confirms that the RLC complex is composed of TRBP (pink star), an 11-kDa RNA fragment (yellow circle), Dicer, and Ago2 (blue star) subunits. The different heterodimers formed in these experiments are rationalized because the Dicer:TRBP complex forms in solution, whereas in the gas-phase loss of the smallest subunit, TRBP is favored, giving rise to a stripped complex Dicer:Ago2 (25).

RISC-loading activity of the reconstituted RLC. (A) Overview of the RISC-loading process, involving pre-miRNA recognition, dicing, passenger strand removal, and guide strand RNA loading onto Ago2, as well as target RNA slicing. The red asterisk indicates the 5′ end ³²P-labeled RNA strand. (B) RISC-processing assays. Lanes indicate the addition of unlabeled single-stranded 21-nt guide RNA (ss guide RNA), single-stranded 21-nt passenger RNA (ss passenger RNA), or Dmpre-let7 RNA (pre-miRNA) to RLC and 5′ end ³²P-labeled sense strand target RNA, 37 nt-A (first four lanes), or to 5′ end ³²P-labeled antisense target RNA, 37-nt B (last two lanes). Asterisks indicate the slicing product expected for the sense (∗) and antisense (∗∗) targets.