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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01076

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CRM1-dependent, but not ARE-mediated, nuclear export of IFN-1 mRNA

¹ Department of Microbiology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan
² Kazusa DNA Research Institute, Kazusa-kamatari, Chiba 292-0818, Japan

View larger version (32K): [in a new window]   Fig. 1. HIV-1 Rev inhibits the nuclear export of IFN-1 mRNA in a NES-dependent manner. (A) Rev NES-dependent inhibition of IFN-1 gene expression. 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 for 16 hours at 37°C. pCG-HA, the empty parental vector, was included to normalise the amounts of the vector-based expression plasmids. pUC12 was included to give a total DNA amount of 20 µg. 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 Materials and Methods. Values are presented as the percentage IFN-1 concentration relative to that secreted from phuIFN-1 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.) (B) The top western blot shows the plasmid dosage-dependent increase in HIV-1 Rev protein expression. The bottom western blot indicates the comparable amounts of Rev and Rev mutant proteins expressed in HeLa cells transfected with the corresponding expression plasmids (16 µg each). (C) Coexpression of Rev results in a NES-dependent reduction in the cytoplasmic IFN-1 mRNA levels, but an increase in the nuclear IFN-1 mRNA levels. HeLa cells were cotransfected with pCG-HA-Rev, pCG-HA-Rev27-29A or pCG-HA-RevM10, and phuIFN-1. Cytoplasmic and nuclear total RNAs were collected at 32 hours after the DNA addition and subjected to RT-PCR analysis as described in the Materials and Methods. The cDNA synthesis reaction mixtures were appropriately diluted to ensure linear amplification of the cDNAs in the PCR reactions. The ratio of each IFN-1 transcript level normalised to the corresponding GAPDH transcript level in the presence of Rev proteins, to that in the absence of Rev proteins, is shown below each lane. C, mock-transfected control. The sizes of the amplicons of the IFN-1 and GAPDH transcripts are indicated. The autoradiographs shown were obtained after 3 hours of exposure at –70°C. The results are from a representative experiment of three independent experiments.

View larger version (48K): [in a new window]   Fig. 2. LMB inhibits the nuclear export of IFN-1 mRNA. HeLa cell nuclei were microinjected with either phuIFN-1, pCRRE or pRSVluc-CTE at 37°C for 30 minutes as described in the Materials and Methods. The cells were further incubated at 37°C for 30 minutes (A,G; t: 0 for cells expressing IFN-1 mRNA and luc-CTE mRNA, respectively) or 45 minutes (D; t: 0 for cells expressing HIV-1 gag mRNA), and then treated for an additional 3 hours with 20 nM LMB (C,F,I; LMB: 3 h) as described in the Materials and Methods. (B,E,H; D10: 3 h) Mock-treated cells. The cells were subsequently fixed, permeabilised and subjected to RNA-FISH as described in the Materials and Methods. Representative examples of each type of injected cell are shown. Scale bar: 10 µm.

View larger version (22K): [in a new window]   Fig. 3. Coexpressed CAN inhibits IFN-1 gene expression in a plasmid dosage-dependent manner. HeLa cells were cotransfected at 37°C for 16 hours with a constant amount of either phuIFN-1 (open circles), pRSVluc-CTE (closed circles) or pCRRE (open triangles), and various amounts of pCG-HA-CAN that are shown on the x-axis. At 32 hours after the DNA addition, cytoplasmic lysates and culture supernatants were obtained to assay the IFN-1 concentrations (open circles) and luciferase activities (closed circles), or to analyse the p55Gag expression by western blotting, as described in the Materials and Methods. The concentrations of IFN-1 and p55Gag, as well as the luciferase activities measured, are given as the percentage protein concentrations or enzymatic activities relative to those from phuIFN-1, pCRRE or pRSVluc-CTE 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.)

View larger version (74K): [in a new window]   Fig. 4. Colocalisation of IFN-1 mRNA with CRM1 in HeLa cell nuclei. HeLa cell nuclei were microinjected with either phuIFN-1 (A-C), pCRRE (D-F) or pRSVluc-CTE (G-I) as described in the legend for Fig. 2. The cells were further incubated for 4 hours at 37°C, and then fixed, permeabilised and subjected to RNA-FISH (A,D,G) and immunochemistry (B,E,H) as described in the Materials and Methods. The mRNAs and CRM1 immunofluorescence images were merged electronically (C,F,I). Representative examples of each type of injected cell are shown. Scale bar: 10 µm.

View larger version (50K): [in a new window]   Fig. 5. IFN-1 mRNA is not colocalised with TAP in HeLa cell nuclei. HeLa cell nuclei were microinjected with either phuIFN-1 (A-C), pCRRE (D-F) or pRSVluc-CTE (G-I) as described in the legend for Fig. 2. The cells were further incubated for 4 hours at 37°C, and then fixed, permeabilised and subjected to RNA-FISH (A,D,G) and immunochemistry (B,E,H) as described in the Materials and Methods. The mRNAs and TAP immunofluorescence images were merged electronically (C,F,I). Representative examples of each type of injected cell are shown. Scale bar: 10 µm.

View larger version (33K): [in a new window]   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.)