Drosophila GW182 and its effector domains decrease stability of tethered mRNA. Northern blotting was used to estimate the levels of firefly and Renilla luciferase mRNAs from the experiment described in Figure 1. mRNA levels were quantified by PhosphorImaging, and amounts of FLuc-boxB were normalized according to Renilla control and expressed as a percentage of FLuc-boxB level in the presence of NHA-lacZ protein (numbers below the figure represent an average of two experiments). (Lanes 1–6) FLuc-boxB and RLuc RNA levels in the presence of tethered NHA-lacZ, NHA-dGW182, and dGW182 fragments: NHA-1-605, NHA-605-830, NHA-940-1215, and NHA-605-1215, accordingly. (Lanes 7,8) To demonstrate that degradation is the effect of tethering, lanes 7 and 8 show untethered FLuc and RLuc in the presence of either (lane 7) NHA-lacZ or (lane 8) NHA-dGW182. The last lane contains total RNA from untransfected S2 cells (no target control).
