Figure 2

Cellular Dicer associates with the HIV-1 TAR stem and loop structure. A) Biotin labeled TAR-RNA was incubated with one milligram whole cell extracts from 293, CEM, ACH2 and BJAB (lanes 1–4), pulled down with streptavidin beads, washed thrice with TNE-150, resolved by SDS-PAGE and detected for Dicer by western blot. One milligram cell extract incubated without biotin-TAR was used as a control (lanes 5–8) and 30 microgram whole cell extract from 293 and CEM cells were included in the western as a positive control for Dicer detection (lanes 9 and 10). B) Unlabeled wild type (TAR-WT) and mutant TAR (TAR-D) were used in a competition experiment to examine the specificity of Dicer binding to TAR. 293 cell extracts were incubated in the presence (lane 1,3,4) or absence (lane 2) of 1.5 micrograms of biotin-labeled TAR. A 2.4 fold excess of TAR-WT (lane 3) or TAR-D (lane 4) was incubated with two of the samples. Biotin-TAR was pulled down with streptavidin beads and detected for Dicer by western blot as in panel A. Densitometry was performed to determine the relative amount of Dicer present after the pulldown, as compared to the positive control (lane 2). C) Pulldown was performed as in panel A, but included a biotin-PolyU and TAR-D in addition to the WT-TAR. Percentages indicate the relative pulldown as compared to TAR-WT.