Figure 3.

Effects of the addition of TPR proteins on RISC assembly. (A) Effect of the addition of TPR proteins on the generation of ss siRNA. AGO1 and non-tagged TPR proteins were synthesized in BYL, mixed, and incubated with gf698-22 siRNA duplex containing the ³²P-labelled guide strand at 25°C for 30 min. RNA was extracted from the reaction mixtures and analysed by 15% native PAGE (left panel). Negative control reactions using mock-translated BYL were performed in parallel. The ss siRNA generation activity was calculated by dividing the intensity of ss siRNA band by the sum of intensities of ss and duplex siRNA bands. The graph shows the averages and s.d. of the relative ss siRNA generation values (‘AGO1+mock’=100%) obtained in three independent experiments (right panel). Different letters indicate statistically significant differences (Student’s t-test, P<0.01). (B) Effect of the addition of TPR proteins on target cleavage activity. AGO1 and TPR proteins were synthesized in BYL, mixed, incubated with gf698-22 siRNA duplex at 25°C for 30 min, and further incubated with ³²P-labelled GF-s target RNA. RNA was extracted and analysed by denaturing 5% PAGE (left panel). To calculate relative target cleavage activity, the intensity of the full-length GF-s target RNA and the 5′ cleavage product bands was quantified. Because full-length GF-s and the 5′ product contain 104 and 86 cytosine residues, respectively, the intensity of the 5′ product bands was multiplied by 104/86 (∼1.21), and divided by the sum of intensity of full-length GF-s and that of the 5′ product multiplied by 1.21. The graph shows the averages and s.d. of the relative target cleavage activity values (‘AGO1+mock’=100%) obtained in three independent experiments (right panel). Different letters indicate statistically significant differences (Student’s t-test, P<0.01). (C) Effect of the addition of TPR proteins on the amount of ss and ds small RNAs copurified with AGO1. FLAG–AGO1 and non-tagged TPR proteins were synthesized in BYL, mixed, and incubated at 25°C for 2, 10, and 30 min with gf698-22 siRNA duplex containing the ³²P-labelled guide strand. FLAG–AGO1 was immunopurified with anti-FLAG antibodies and copurified RNA was analysed by 15% native PAGE. The intensity of copurified ds and ss siRNA bands was measured, and the relative amount (‘AGO1+mock’=100%) was shown in the left graph. Experiments were repeated three times, and the representative data were shown.