Ago2 bound to multiple nonoverlapping GW-rich regions of GW182. (A) GW182 fragments coprecipitated with C-terminal half of Ago2. GST-PIWI (“aa478-860”) was cotransfected with GFP-tagged GW1Δ1 (lane 1), GW1Δ1a (lane 2), GW1Δ1b (lane 3), GW1Δ7 (lane 4), TNR (lane 11), or GW1Δ5 (lane 12) into HeLa cells. Similar to positive control GW1Δ1 (lane 6), GW1Δ1a (lane 7), GW1Δ1b (lane 8), GW1Δ7 (lane 9), GFP-GW1Δ5 (lane 14), but no GFP-TNR (lane 13), were detected in GST-PIWI precipitates. GST-tagged N1, N-terminal fragment from an unrelated protein hZW10, was cotransfected with GFP-GW1Δ1 (lane 5) as a negative control and no interaction was detected (lane 10). Asterisks indicate the corresponding GFP-tagged constructs in Western blot. (B) GW182 fragment GW1Δ12 coprecipitated with Ago2. Flag-Ago2 was cotransfected with GST-tagged TNR (lanes 1,3) or with GW1Δ12 (lanes 2,4), which contains the conserved sequence for Ago2 interaction. Flag-Ago2 was coprecipitated with GST-tagged GW1Δ12 but not TNR.
