Dynein Associates with oskar mRNPs and Is Required For Their Efficient Net Plus-End Localization in Drosophila Oocytes
Figure 1
The Dynein complex associates with oskar mRNA in vivo.
(A) Ovarian lysates from wild-type flies were subjected to immunoprecipitation using the following antibodies; a goat anti-mouse control antibody (lane 2), Khc (lane 3), Dhc (lane 4), Glued/p150 (lane 5), and Lis-1 (lane 6). The co-precipitating RNAs were extracted and analyzed using RT-PCR. A specific enrichment of oskar mRNA could be detected in the Glued pellet. A small but significant enrichment of bicoid mRNA could also be detected in the Glued pellet. Lane 1 represents total RNA from wild-type flies analyzed using primers against oskar, bicoid and gurken. This experiment was repeated three times.
(B) Ovarian lysates from wild-type flies and flies expressing Dic-GFP were subjected to immunoprecipitation using GFP antibody beads. The co-precipitating RNAs were extracted and analyzed as in panel A (lanes 3 and 4). oskar, bicoid and gurken mRNAs were specifically enriched in the Dic-GFP pellet. Total RNA from the same flies was also analyzing using RT-PCR with primers against oskar, bicoid and gurken (lanes 1 and 2). The IP RT-PCR experiment was repeated four times.
(C) Ovarian lysates from wild-type flies and flies expressing Dic-GFP were subjected to immunoprecipitation using GFP antibody beads. The precipitates were run on gel and analyzed by western blotting using the indicated antibodies (lanes 3 and 4). The total fraction from both lysates were similarly analyzed (lanes 1 and 2). As expected, Dhc and Glued co-precipitated with Dic-GFP. In addition, a small enrichment of Khc was also detected in the Dic-GFP pellet. This experiment was repeated three times.