Gene silencing mediated by tethered C-terminal half of Ago2 required GW182. (A) Tethered PIWI (“aa478-860”) down-regulated protein synthesis to the same extends of other Ago proteins. HeLa cells were transfected with constructs expressing the RL-5BoxB reporter, control FL reporter, and indicated NHA-tagged proteins. Bar graphs represent normalized mean values of RL/FL activities with standard errors. The RL/FL values in cells with tethered NHA-tagged Ago2, PIWI, Ago1, and Ago4 were significantly reduced compared with the value in NHA only group, which was normalized as 1. The NHA tagged PAZ, Ago3m, or HA-Ago2 did not show repression effect on the reporters. The expression of fusion proteins were determined by Western Blot using anti-HA mAb and are indicated below the bar graphs. The assay was repeated a minimum of three times. Asterisks indicate groups have significant difference compared with the NHA only group (unpaired t-test, P < 0.0001). No significant difference was shown between any two groups with asterisk (unpaired t-test, P > 0.05). (B) Translational repression mediated by tethered Ago2 or PIWI (“aa478-860”) was greatly impaired upon GW182 knockdown. HeLa cells were transfected with siRNA for either GW182 (siGW182) or GFP (siGFP). Thirty hours later, cells were transfected again with constructs expressing reporter RL-5BoxB, control FL reporter, and the indicated NHA-tagged proteins. Bar graphs represent the reduction of RL/FL in cells with tethered NHA-Ago2 or NHA-PIWI compared with those in cells with tethered NHA. The reduced values of RL/FL in cell transfected with siGFP were set as 1. Error bars indicate standard errors. The assay was performed in triplicate and was repeated two times. *Significant difference (unpaired t-test, P < 0.01). (C) GW182 knockdown by siRNA was confirmed by quantitative real-time PCR. The bar graphs represent normalized mRNA level of GW182 with standard errors. The mRNA level of GW182 in cells transfected with GFP-siRNA was set as 1. The experiment was performed in triplicate.
