TNRC6A, TNRC6B, and TNRC6C interact with human AGO1–AGO4. (A) Domain organization of human TNRC6A–TNRC6C and D. melanogaster GW182. N-GW, M-GW, and C-GW: N-terminal, middle, and C-terminal GW-repeat-containing regions, respectively (the number of GW-repeats for each region is indicated in brackets). The white sectors within the N-terminal regions do not contain GW-repeats. UBA (yellow box): ubiquitin associated-like domain; Q-rich (cyan box): region rich in glutamine; RRM (green box): RNA recognition motif. Red boxes I and II: two conserved motifs within the N-terminal GW repeats. (Orange box) Ago-hook motif; (gray box) conserved motif III in the middle region. Numbers underneath the protein outline represent amino acid positions at fragment boundaries for each protein. Black arrows indicate the boundaries of the N and C-terminal protein fragments used in this study. The protein domains sufficient for the interaction with AGO1 and for silencing are indicated. (B–E) GFP-AGO1–AGO4 and HA-TNRC6A–TNRC6C were coexpressed in human HEK293 cells as indicated. Cell lysates were immunoprecipitated using anti-GFP antibodies. GFP-MBP served as a negative control. Inputs (2%) and immunoprecipitates (30%) were analyzed by Western blotting using anti-GFP or anti-HA antibodies.
