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First published online 29 June 2004
doi: 10.1242/jcs.01129

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Nucleolar localization and mobility analysis of the NF-B repressing factor NRF

Ina Niedick, Natali Froese, André Oumard, Peter P. Mueller, Mahtab Nourbakhsh^*, Hansjörg Hauser and Mario Köster

Department of Gene Regulation and Differentiation, GBF – German Research Center for Biotechnology, Mascheroder Weg 1, 38124 Braunschweig, Germany

View larger version (37K): [in a new window]   Fig. 1. NRF domains and DNA fragments used in the study (A) Amino acid sequence of human NRF as deduced from the cDNA clone used in this study. Asterisk indicates the stop codon. (B) Schematic illustration of the NRF protein showing the NLS, the NF-B repression domain, the DNA binding domain (DBD) and consensus sequence motifs for RNA binding (JAG), nucleic acid binding (G-Patch) and single-stranded nucleic acid binding (R3H). Several dsRNA binding motifs are found between amino acid (aa) 349 and aa 512 (dashed line). (C) Schematic representation of the full-length NRF tagged with His-Myc epitope or with GFP and NRF deletion mutants fused to GFP. The His-Myc-tag was fused in-frame to the amino terminus of NRF, while GFP was added in-frame to its C-terminal end.

View larger version (96K): [in a new window]   Fig. 2. dsRNA binding activity of NRF. Uniformly 32P-labeled 469 nt IFN- T7-derived RNA (see Methods section) was incubated with in vitro produced full-length NRF fusion protein or the indicated deletion mutants. The reaction mixture was subjected to electrophoresis on a native gel to separate RNA-protein complexes from the free probe. The arrows indicate the migration of IFN- RNA as a free probe and as complexes with NRF and PKR. The autoradiogram shows free and bound RNA. Addition of antibody directed against the Myc-tag (that specifically inhibits the complex formation between His-Myc-NRF and IFN-) is indicated.

View larger version (65K): [in a new window]   Fig. 3. Localization of full-length and mutant NRF in interphase nuclei. (A) Fluorescence microscopy images (b,d) and the corresponding phase-contrast images (a,c) of C243 cells expressing His-Myc-tagged NRF (a,b) or NRF-GFP fusion protein (c,d). To visualize the cytoplasmic and nucleoplasmic presence of NRF, confocal laser scanning microscopy of GFP-tagged full-length protein was carried out under non-saturating (e) and saturating (f) imaging conditions in living C243 cells. (B) C243 cells expressing full-length NRF-GFP (a), NRF362-690-GFP (b) or the nucleolic marker protein GFP-hfbr2_82i24 (d) were treated with 5 µg/ml actinomycin D. Intracellular localization of the GFP-tagged proteins was determined by confocal laser scanning microscopy in living cells. Representative images from cells after 8 hours of actinomycin D treatment are shown. As non-treated control GFP-hfbr2_82i24 is shown in c. (C) C243 cells expressing His-Myc-tagged NRF were either left untreated or treated with 5 µg/ml actinomycin D for 8 hours. Nuclear and nucleolar fractions were prepared as described under Materials and Methods. Equal amounts of protein were resolved by SDS-PAGE, blotted onto a nitrocellulose membrane and probed with antibody directed against the Myc-tag. (D) NRF deletion mutants fused to GFP were expressed in C243 cells. The subcellular localization of the various mutant proteins was determined by confocal laser scanning microscopy in living cells. (a) NRF1-550-GFP, (b) NRF1-480-GFP, (c) NRF1-434-GFP, (d) NRF1-402-GFP, (e) NRF 1-361-GFP, (f) NRF362-690-GFP.

View larger version (77K): [in a new window]   Fig. 4. Dynamics of nucleolar targeting of NRF-GFP. (A) C243 cells stably transfected with NRF-GFP were subjected to FRAP analysis. The area of an entire nucleolus was bleached (indicated by an arrow in the first post-bleach panel) and images collected before and at the indicated time points after the end of the bleach pulse are shown. (B) Quantitative data of fluorescence recovery kinetics for NRF-GFP were recorded and plotted over time. The fluorescence intensities in bleached and unbleached nucleoli were measured for two adjacent cells (shown below; the bleached nucleolus is indicated by an arrow in the pre-bleach panel). Plotted data were not corrected for the overall loss of fluorescence induced by the image collection, to allow a quantitative comparison of signal loss in unbleached areas with signal gain in the bleached area. The FRAP rate of the bleached nucleolus (number 1) is represented by red diamonds; 2 (green squares) and 3 (blue triangles) are unbleached nucleoli of the same cell; 4 (orange circles) and 5 (magenta squares) are nucleoli of an adjacent cell. (C) C243 cells expressing NRF1-361-GFP were subjected to FRAP analysis. The area of an entire nucleolus was bleached (indicated by an arrow in the first post-bleach panel) and images collected before and at the indicated time points after the end of the bleach pulse are shown. (D) Quantitative data of fluorescence recovery kinetics for NRF1-361-GFP were recorded and plotted over time. Fluorescence intensities of bleached and unbleached nucleoli as well as in nucleoplasmic areas were determined. The FRAP rate of the bleached nucleolus (1) is indicated by red diamonds; 2 (green squares) and 3 (blue triangles) are unbleached nucleoli of the same cell; 4 (orange circles) is the nucleoplasm.

View larger version (44K): [in a new window]   Fig. 5. FLIP analysis of NRF-GFP. C243 cells were stably transfected with NRF-GFP and subjected to FLIP analysis. The bleached regions in the cytoplasm (A) or in the nucleoplasm (B) are indicated with white rectangles and fluorescence intensity is shown in false color code. Each image series shows the fluorescence prior to bleaching (0 seconds) and after three consecutive bleaching periods of the indicated time (34 seconds for A; 27 seconds for B).

View larger version (55K): [in a new window]   Fig. 6. Quantitative FRAP analysis of full-length and mutant NRF-GFP. (A) C243 cells stably transfected with expression plasmids encoding the full-length NRF-GFP were subjected to quantitative FRAP analysis. The area of an entire nucleolus was bleached and fluorescence recovery kinetics for the bleached region and change of fluorescence intensity in an unbleached nucleolus were measured. To allow a quantitative comparison of signal loss in unbleached areas with signal gain in the bleached area, plotted data were not corrected for the overall loss of fluorescence induced by the measurements of fluorescence intensity. For clarity, only selected time points are marked by a symbol. Open symbols indicate the bleached area, whereas closed symbols mark the area of unbleached nucleoli. Each graph represents the average of data from ten single cells. (a) Fluorescence recovery kinetics for NRF-GFP were compared in C243 cells (squares) and in a stably transfected NIH3T3 cell line (triangles). (b) Fluorescence recovery kinetics for NRF-GFP at room temperature (squares) and at 37°C (triangles) were compared in C243 cells. (c) One part of a nucleolus was bleached and fluorescence recovery kinetics for the bleached region (open squares) and change of fluorescence intensity in the unbleached area (closed circle) of the same nucleolus were measured. (B) Kinetics of fluorescence recovery for NRF-GFP and NRF362-690-GFP were measured both in the nucleoplasm (a,b) and in the cytoplasm (c,d). Bleaching was done in a circular area and fluorescence recovery for the bleached region and change of fluorescence intensity in an unbleached area of the same size were plotted over time. For clarity, the scale of the y-axis was changed. (a,c) NRF-GFP; (b,d) NRF362-690-GFP. (C) C243 cells transfected with expression plasmids for the indicated GFP-tagged NRF deletion mutants were subjected to quantitative FRAP analysis. The overall FRAP settings are described in (A). (a) NRF1-550-GFP, (b) NRF1-480-GFP, (c) NRF1-434-GFP, (d) NRF1-402-GFP, (e) NRF1-361-GFP, (f) FRAP analysis of NRF1-361-GFP in the nucleoplasm (np), (g) NRF362-690-GFP, (h) Comparison of the FRAP kinetics for full-length NRF and the indicated deletion mutants.

View larger version (18K): [in a new window]   Fig. 7. Quantitative FRAP analysis of NRF-GFP during viral infection. C243 cells expressing full-length NRF-GFP or the nucleolus marker protein GFP-hfbr2_82i24 were infected with Sendai virus or Newcastle disease virus (NDV). Before and at the indicated time points post infection cells were subjected to FRAP analysis as described in Fig. 6A. Inter-nucleolic mobility of NRF-GFP was measured. For clarity, only fluorescence recovery kinetics for the bleached nucleoli are shown. The change of fluorescence intensity in the unbleached nucleoli were also determined and do not differ from the changes presented in Fig. 6A. Each individual time point represents the average of data from five single cells. (A) NRF-GFP-expressing cells infected with Sendai virus. (B) NRF-GFP-expressing cells infected with NDV. (C) GFP-hfbr2_82i24-expressing cells infected with Sendai virus.