FIG. 6.
Protein A induces RNA1fs membrane association in Drosophila. D. melanogaster cells expressing RNA1fs alone or together with protein AD692E were collected 2 days after transfection. (A) Flotation and RNA extraction were done as described in the legend to Fig. 4. One-step real-time RT-PCR was performed on 5 ng of RNA, using primers and a probe specific for either RNA1 or actin mRNA. The RNA1fs amount in each gradient fraction, determined from four independent experiments, was graphed as the percentage of RNA1fs in the entire gradient. (B) Total RNA was extracted and analyzed by Northern blotting as described in the legend to Fig. 1 or by real-time RT-PCR as described above. Data from at least three independent experiments are shown. (C) Protein and RNA were extracted from equal cell volumes of yeast or Drosophila expressing RNA1fs and protein AD692E and were analyzed by Western blotting for protein A as described in the legend to Fig. 1 and by real-time RT-PCR for positive-strand RNA1 as described above, except that 75 ng of total RNA was used. The ratios of protein A Boehringer light units to μg of total protein (Ptn A/total Ptn), positive-strand RNA1 molecules per ng of RNA loaded to ng of total RNA (RNA1/total RNA), and protein A/total protein to RNA1/total RNA (Ptn A/RNA1) are graphed on two different scales. Averages from three independent experiments are shown.