Functional interaction between Crm1 and K-Rev. (A) Microaffinity columns containing GST (lane 1), GST-Crm1 only (lane 2), GST-Crm1 preincubated with Ran-GTP (lane 3), or GST-Crm1 preincubated with Ran-GDP (lane 4) were prepared as described (6, 28). Equal aliquots of ³⁵S-labeled K-Rev were then loaded onto each column, and bound proteins were eluted by using 0.05% SDS. The eluates were resolved by gel electrophoresis and visualized by fluorography. (B) Human 293T cells (35-mm cultures) were transfected with 500 ng of pBC12/CMV/K-Rev and 1 μg of pBC12/CMV/ΔCAN or the parental pBC12/CMV plasmid by using the calcium phosphate procedure. At ≈48 h after transfection, the cells were fixed and stained as described (6) by using a 1:500 dilution of a rabbit polyclonal anti-K-Rev antiserum and a 1:2,000 dilution of a FITC-conjugated donkey anti-rabbit antiserum (The Jackson Laboratory). No staining was observed in mock-transfected cultures. (Bar ≈20 μm.)

Direct interaction between recombinant K-Rev and the K-RRE in vitro. (A) The RNA gel-shift procedure used has been described (25). All reactions contained 1 μg of yeast tRNA and 4 μg of rRNA as nonspecific competitors. A ³²P-labeled 434-nt K-RRE probe or a 293-nt V-RRE probe was prepared by in vitro transcription, and ≈1 ng (4 × 10⁴ cpm) of probe was then added to a premade in vitro reaction containing no (lanes 1 and 6, Neg.), ≈3 ng (lanes 2 and 7), ≈9 ng (lanes 3 and 8), ≈27 ng (lanes 4 and 9), or ≈81 ng (lanes 5 and 10) of GST-K-Rev. After incubation for 10 min at 4°C, reaction products were resolved on a 5% native polyacrylamide gel and visualized by autoradiography. C1 and C2 indicate distinct protein complexes. (B) The procedure was performed as described for A, except that the reaction mix, containing 9 ng of GST-K-Rev, was preincubated for 10 min at 4°C with an unlabeled K-RRE or H-RRE competitor RNA. (lane 1, Neg.) No added GST-K-Rev; (lane 2) no additional RNA competitor; (lanes 3 and 7) ≈3 ng of RNA competitor; (lanes 4 and 8) ≈12 ng of RNA competitor; (lanes 5 and 9) ≈50 ng of RNA competitor; (lanes 6 and 10) ≈200 ng of RNA competitor.

K-Rev activates the cytoplasmic expression of an unspliced mRNA bearing the K-RRE. (A) Cells (293T) were cotransfected with a pDM128-based indicator construct bearing the K-RRE, H-RRE, or V-RRE, together with a pBC12/CMV-based expression vector encoding K-Rev, H-Rev, or V-Rev or with pBC12/CMV as a negative (Neg) control. pBC12/CMV/β-GAL was cotransfected as an internal control. At ≈48 h after transfection, cells were harvested, and induced CAT and β-GAL activities were determined. The presented data were corrected for minor variations in β-GAL activity and are representative of four independent experiments. (B) Cells (293T) were transfected with pDM128/K-RRE together with either pBC12/CMV/K-Rev (+) or the parental pBC12/CMV plasmid (−). At ≈48 h after transfection, nuclear (N) and cytoplasmic (C) RNA fractions were isolated and analyzed by RNase protection assay by using a previously described (9) ³²P-labeled RNA probe that traverses the 3′ splice site. Lane 5 contains total (T) RNA from mock-transfected 293T cells. U, unspliced RNA; S, spliced RNA. (C) Cells (293T) were transfected with pDM128/K-RRE and pBC12/CMV (Neg), pBC12/CMV/K-Rev, or pBC12/CMV/H-Rev, as described for A, except that an additional 500 ng of either pBC12/CMV/ΔCAN or pBC12/CMV was cotransfected. The pDM128/CTE indicator plasmid contains the MPMV CTE. Induced CAT and β-GAL activities were determined as described for A. ΔCAN does not significantly affect expression of the β-GAL internal control (6). These data are representative of three independent experiments.