FIGURE 3.

Formation of the A3G:NC complex is mediated by unstructured RNAs. (A) The purified A3G-Myc-His was untreated (lanes 3,4) or had been incubated with RNase A (all other lanes). Input A3G-Myc-His protein (5% of total) is shown in lanes 1 and 2. A3G-Myc-His protein was incubated with purified GST (lane 3) or GST-NC (lanes 4–10). In lanes 6–10, yeast RNA was added to the incubation: lane 6, 1.25 μg; lane 7, 2.5 μg; lane 8, 5 μg; lane 9, 10 μg; lane 10, 20 μg. A3G:NC complexes were recovered using glutathione-agarose beads and bound A3G-Myc-His protein visualized by Western analysis using a rabbit polyclonal anti-A3G antiserum (upper panel). Input GST or GST-NC protein was also analyzed by Western blot (lower panel). (B) Similar to panel A, except that the added RNAs (10 μg in each case) represent yeast total RNA (lane 3); purified yeast tRNA (lane 4); total human cell RNA (lane 5), or total human RNA that had been subjected to one round (lane 6) or two rounds (lane 7) of purification on an oligo-dT column. This experiment used exclusively RNase A treated A3G-Myc-His protein. (C) Ethidium bromide-stained agarose gel visualizing 5 μg of each of the RNA samples used in panel B. As may be seen, the yeast RNA sample (Ambion) in lane 1 is degraded to the point where it is smaller than the 70–80-nt-long yeast tRNAs shown in lane 2. Lanes 3–5 reveal the removal of human rRNA as the total human RNA sample (lane 3) was subjected to one (lane 4) or two (lane 5) rounds of oligo-dT purification. (D) Similar to panels A and B, except that this experiment also analyzed in vitro transcribed human Y4 RNA and 7SL RNA.