Identifying the interaction of C-terminal half of Ago2 with GW182 fragments using GST pull-down assays. (A) Schematic of human GW182 and Ago2 deletion constructs used in this study. Amino acid residues of GW182 constructs are referenced to the TNGW1, the longer isoform of GW182 (GenBank Accession NM_014494.2). Q-repeat, glutamine repeat (box in white); Q/N-rich, glutamine/asparagine-rich region (box in magenta); RRM, RNA recognition motif (box in green); GW-rich, glycine/tryptophan-rich region (boxes in yellow); N-GW, N-terminal GW-rich region; M-GW, middle GW-rich region; C-GW, C-terminal GW-rich region. Human Ago2 contains two conserved domains: PAZ domain (box in blue) and PIWI domain (box in red). (B) Endogenous Ago2 coprecipitated with GW182 fragments GW1Δ1 (“aa254-751”) and GW1Δ10 (“aa566-1343”) but not with TNR (“aa1-204”). GST-tagged TNR (lanes 1,4), GW1Δ1 (lanes 2,5), or GW1Δ10 (lanes 3,6) was transfected into HeLa cells. Endogenous Ago2 was detected by using rabbit polyclonal anti-Ago2, which also contained anti-GST activity. In a longer exposure (lanes 7–9), Ago2 was detected more clearly being coprecipitated with GST-GW1Δ10 (lane 9) but still absent in GST-TNR precipitates (lane 7). (C) GW182 fragments GW1Δ1 and GW1Δ10 coprecipitated with Ago2 fragment PIWI (“aa478-860”) but not with PAZ (“aa1-480”). GST-GW1Δ10 (lanes 1–6) or GST-GW1Δ1 (lanes 7–10) was cotransfected with Flag-tagged Ago2, PIWI, or PAZ. Full-length Ago2 (lanes 4,9) and PIWI (lanes 5,10), but not PAZ (lane 6), coprecipitated with GST-GW1Δ10 or GST-GW1Δ1. (D) GW182 fragment TNR did not pull down Ago2. Flag-Ago2 was cotransfected with GST-tagged TNR (“aa1-204,” lanes 1,3) or with GW1Δ1 (lanes 2,4) as a positive control.
