FIGURE 4:

miRNP retention in polysomal pool precludes its RISC activity. (A) Assay to measure RISC cleavage activity of polysomal miRISC-122 isolated from LDC and HDC HeLa cells cotransfected with FLAG-HA-AGO2 and miR-122 expression plasmid. Normalization of RISC activity was done against the HA-AGO2 detected by Western blot analysis. (B) Relative specific activity of polysomal miRISC-122 isolated from HDC and LDC states with respect to AGO2 protein levels (left axis) or AGO2-associated miR-122 content (right axis). (C) In vitro translation activates polysomal miRISC. Polysomes isolated from HDC HeLa cells transfected with miR-122 expression plasmid was subjected to in vitro translation in rabbit reticulocyte lysates for 1 h at 30 and 0°C (control) before measuring the RISC activity at 30°C. Left, autoradiogram showing the end product of the reaction analyzed on a denaturing PAGE. Arrowhead marks the cleaved RNA bands. Right, relative RISC activity. (D) Distribution of FH-AGO2 on a 30–55% sucrose gradient after isolated polysomes from HDC HeLa cells were incubated with rabbit reticulocyte lysates at 30°C for 1 h. FH-AGO2 distribution at 0°C served as control. Top, FH-AGO2 detected by Western blot with anti-HA antibody. Bottom, quantification of the AGO2 levels in different fractions done by measuring the band intensity. (E) Binding of AGO2 to new target RNA is reciprocal to its association with polysomes. Top, scheme of the experiment. Bottom, relative amount of RL-3xbulge-miR-122 mRNA bound to FH-AGO2 after the polysomal FH-AGO2 was subjected to translation reaction at 37 or 0°C. ns, nonsignificant, *p < 0.05, **p < 0.01, and ***p < 0.0001. The p values were determined by paired t test. All experiments were performed a minimum of three times.