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Fig. 6. 3' UTR truncation does not negatively affect the nuclear export of IFN-
1 mRNA. (A) HeLa cell nuclei were microinjected with either phuIFN-1 or phuIFN-1/
3' UTR at 37°C for 30 minutes. The cells were further incubated at 37°C for 4 hours and then fixed, permeabilised and subjected to RNA-FISH as described in the legend for Fig. 2. Upon image analysis of full length IFN-1 mRNA- or 3' UTR-truncated IFN-1 mRNA-expressing cells, the cytoplasmic versus nuclear ratios of each IFN-1 mRNA signal were determined and the total cellular mRNA signals were quantified as described in the Materials and Methods. The mean±s.e.m. values obtained from a representative experiment are shown. For the analyses, 25-50 cells were analysed on one Cellocate coverslip and three coverslips were examined for each plasmid; each experiment was repeated three times. The structure of the full length IFN-1 gene (nt 1-876) expression vector is depicted at the top left. The arrow, open circle and star show the transcription start site, and translation start and stop codons, respectively. Nt 1-67, 68-637 and 638-876 are the 5' UTR, coding region and 3' UTR, respectively. The ARE present in the 3' UTR is also shown. (B) Representative images used for the analyses are shown. 1-876: phuIFN-1-injected; 1-637: phuIFN-1/3' UTR-injected cells. (C) Rev NES-dependent inhibition of the expression of the 3' UTR-truncated IFN-1 gene. HeLa cells were cotransfected with various amounts of either pCG-HA-Rev (open circles), pCG-HA-Rev27-29A (closed circles) or pCG-HA-RevM10 (open triangles), and 1 µg of phuIFN-1/3' UTR as described in the legend for Fig. 1A. At 32 hours after the DNA addition, the culture supernatants were collected to quantify the IFN-1 secreted from the transfected cells as described in the legend for Fig. 1A. Values are presented as the percentage IFN-1 concentrations relative to that secreted by phuIFN-1/3' UTR alone transfected HeLa cells. Values of a representative experiment of three independent transfection experiments are shown; mean±s.e.m. of triplicate samples. (Bars cannot be seen when they are smaller than the graph symbols.)
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