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. 2005 Dec 1;19(23):2837–2848. doi: 10.1101/gad.1370605

Figure 3.

Figure 3.

Recombinant Drosophila full-length AGO1 and the PIWI domains of AGO1 and AGO2 cleave target RNA, depending on the small RNA sequences with which they are associated. (A) luc target RNA was efficiently cleaved by GST-AGO1 associated with luc guide siRNA. Purified GST-AGO1 (Supplementary Fig. S3) was first incubated with luc guide siRNA and then luc target RNA (luc180) radiolabeled at the 5′-end was added. Addition to the reaction of EDTA at 10 mM abolished the activity. Without siRNA addition, the target RNA cleavage is not observed. GST itself does not show the activity. (B) A similar experiment to A was carried out using miRNA let-7 (miR-let-7) and the miR-let-7 target RNA (Okamura et al. 2004). Target RNA is cleaved in an AGO1-miR-let-7-dependent manner. (C) Reconstitution of Slicer activity with the PIWI domain of AGO2. All of the recombinants used were first incubated with luc guide siRNA and then luc target RNA (luc180) radiolabeled at the 5′-end was added and incubated. GST-AGO2-PIWI-I and GST-AGO2-PIWI-G cover the PIWI domain of AGO2, but GST-AGO2-PIWI-D contains only a portion of the PIWI domain as indicated below. (D) The PIWI domain of AGO1 preassociated with guide siRNA also shows activity to cleave target RNA.