Figure 1.

Reconstruction of the export of the singly spliced transcript in Xenopus oocytes. (A) Gene expression of HIV-1 is regulated by RNA export. (B) Schematic representation of pre-ftzRRE RNA used for the export analysis. (C) ³²P-labeled pre-ftzRRE RNA was microinjected into the nucleus of Xenopus oocytes together with ³²P-labeled U1ftz (elongated U1), U6RRE and U6Δss, in either the absence (lanes 1–8) or presence (lanes 9–14) of the purified recombinant Rev protein (160 fmol/oocyte), with either CTE (50 fmol/oocyte; lanes 5, 6, 11 and 12) or the CTE mutant M36, which does not bind TAP/NXF1 (CTEmut, 50 fmol/oocyte; lanes 7, 8, 13 and 14), or without the inhibitor (lanes 1–4, 9 and 10). RNA was extracted from nuclear (N) and cytoplasmic (C) fractions, immediately (0 h; lanes 1 and 2) or 1 h (1 h; lanes 3–14) after the injection, and were analyzed using 6% denaturing PAGE. (D) The same experiments as in (C) were performed in the absence (lanes 1–6) or presence (lanes 7–10) of Rev, except with BSA-NES (190 ng/oocyte; lanes 3, 4, 7 and 8) or BSA-mut (M10) NES, which does not bind CRM1 (190 ng/oocyte; lanes 5, 6, 9 and 10), or without the inhibitor (lanes 1 and 2). RNA export was analyzed immediately (0 h; lanes 1 and 2) or 1 h (1 h; lanes 3–10) after the injection. (E) Quantitation of the export of spliced ftzRRE RNA from three independent experiments performed as in (C) and (D). Averages and standard deviations with CTE (gray bars) or BSA-NES (black bars), or without inhibitors (none; white bars) in the absence (-) or presence (+Rev) of Rev are shown.