Figure 3

Dicer cleaves the HIV-1 TAR structure in vitro. A) In vitro transcribed HIV-1 TAR was incubated overnight in the presence of recombinant Dicer or heat-inactived (95° for two minutes) recombinant Dicer, separated on a 6% TBE-UREA gel and stained with ethidium bromide. A 100 bp DNA ladder (lane 1 – band visible at the top of the gel corresponds to 100 bp) and commercially available siRNA against siEGFP (lane 2) were included as size standards. Lane 3 – one microgram in vitro transcribed TAR. Lane 4 – one microgram TAR incubated with rDicer. Lane 5 – one microgram TAR incubated with heat inactivated Dicer. Arrow indicates the ~21 nt band generated by Dicer cleavage. Densitometry indicates the signal in the 21 nt region of the gel relative to undigested TAR. B) Digest was repeated as in panel A. Products were resolved on a 15% TBE-Urea gel and visualized by autoradiography. MW (lane 1) was exposed for 5 minutes, while the digests (lanes 2–4) were exposed for 2 hours. Numbers to the left indicate the size of the associated RNA marker. C) Predicted structures of the TAR mutants used for Dicer cleavage analysis. TAR-A is a compensatory mutant, where the base pairs in the stem have been switched. TAR-B has the bulge (TAR binding site) deleted. TAR-C has a deletion in the terminal loop. TAR-D has a truncated stem. TAR-E has a shortened stem and a long 5' tail, or may fold into a two looped structure. D) TAR mutants pictured in panel B were digested with Dicer, separated on a TBE-UREA gel and stained with ethidium bromide. Lane 1 – 100 bp marker. Pictured are mutants TAR elements before and after Dicer treatment; TAR-A (lanes 2 and 3), TAR-B (lanes 4 and 5), TAR-C (lanes 6 and 7) and TAR-D (lanes 8 and 9). Densitometry was performed as in panel A, where the intensity of the 21 nt region of the gel is shown in relation to the undigested mutant.