FIGURE 4.

Target RNA binding and silencing are affected by mutation of the N-PAZ lobe. A, schematic of target RNA binding. Drosophila Argonaute variants were preincubated with saturating amounts of guide RNA and radiolabeled target RNA was added to the complex. B, target binding of DmAgo1 with bantam versus miR-279. DmAgo1 bound to either bantam or miR-279 was titrated in filter binding assay with radiolabeled 43-nt target RNA, which base pairs with positions 1–12 of bantam. Binding was performed in the absence of magnesium to prevent cleavage. C, tethered luciferase reporter assay for evaluating silencing activity of Argonaute variants. DmAgo1 variants and GW182 were co-expressed as λN fusion proteins in S2 cells with a luciferase reporter containing box B elements in the 3′-UTR. DmAgo1-YK and GW182 served as negative and positive controls, respectively. D, Western blot analysis of immunoprecipitated DmAgo1 variants and co-purified GW182 protein. FLAG-tagged DmAgo1 variants were expressed, immunoprecipitated, and then probed using anti-FLAG antibody. The asterisk indicates a nonspecific band that served as loading control. Levels of co-purified endogenous GW182 were determined using GW182 antibody. E, GFP-tagged GW182 and FLAG-tagged DmAgo1 variants were co-expressed in S2 cells. Anti-FLAG antibody was used to pull-down the Argonaute variant, and Western blot analysis was performed with anti-GFP antibody to determine the extent of association with GW182. CTR, control.