Three separate domains of Drosophila GW182 are sufficient to repress tethered mRNA. (A) Schematic representation of Drosophila GW182 protein and generated dGW182 deletion mutants. The numbers correspond to the amino acid positions. (B) Schematic representation of reporter constructs: FLuc-boxB contains firefly luciferase coding sequence and 3′-UTR with five boxB sites specifically binding to λN peptide; RLuc contains Renilla luciferase coding sequence and no boxB sites (Rehwinkel et al. 2005). (C) Repression of FLuc-boxB mRNA by NHA-dGW182 and its deletion mutants. Drosophila S2 cells were co-transfected with plasmids encoding for FLuc-boxB, RLuc, and full-length NHA-dGW182 or one of the NHA-dGW182 deletion mutants depicted in A. As negative controls, either NHA alone, NHA fused to β-galactosidase (NHA-lacZ), or mutant NHA-Ago1 F594VF629V (designated as 2F2V) not able to recruit dGW182, or HA-dGW182 without λN peptide was transfected instead of NHA-dGW182 or NHA-dGW182 deletions. Cells were lysed 2 or 3 d post-transfection, and protein levels were measured with dual luciferase assay. Expression of firefly luciferase was normalized to that of the Renilla luciferase. Values are presented as a percentage of firefly luciferase produced in the presence of NHA-lacZ. Values represent the average of at least four experiments. The error bar shows the standard deviation. (D) Expression of different HA-fusion proteins was estimated by Western blotting with antibodies directed against HA-peptide.
