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First published online 11 March 2003
doi: 10.1242/jcs.00371

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Dynamic association of RNA-editing enzymes with the nucleolus

¹ Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, 1649-028 Lisbon, Portugal
² MRC Human Genetics Unit, Western General Hospital, Crewe Road, Edinburgh, EH4 2XU, UK
³ Department of Anatomy and Cell Biology, Biomedicine Unit Associated with the CSIC, University of Cantabria, Santander, Spain

View larger version (59K): [in a new window]   Fig. 1. Subcellular distribution of endogenous ADAR1. (A) Schematic representation of hADAR1 proteins (150 kDa and 110 kDa forms). The most important domains of hADAR1 are indicated as follow: ZBDs, Z-DNA-binding domains (light gray); dsRBDs, double-stranded RNA binding domains (black boxes); deaminase domain (dark gray box). The partial ADAR1 fragments (black lines) shown were expressed in E. coli and used for production of rabbit polyclonal antibodies. Numbers denote amino acid positions relative to full-length hADAR1. (B) Western-blot analysis of HeLa and COS7 total cell extracts prepared and fractionated by SDS-PAGE. Western blotting was then performed with a pre-immune serum (lane 1) and with anti-ADAR1 antibodies: 007 (lane 2) and 668 (lane 3-5). Extracts of COS7 cells transiently transfected with Flis-ADAR1 (Fig. 2) are shown in lane 5. All antibodies recognized the full-length hADAR1 (150 kDa) and the 110 kDa form of ADAR1. Molecular weight markers are shown on the left. (C) HeLa cells were immunostained with antibodies 007 and 668, and with the respective pre-immune sera. The panels are representative of the labeling patterns observed. (D) Neurosecretory neurons from the supraoptic nucleus were obtained from squash preparations of rat hypothalamus and analysed by indirect immunofluorescence. The cells were double-labeled with affinity-purified anti-ADAR1 antibody (007) and an anti-histone antibody. The ADAR1 antibody produces a non-homogeneous nucleoplasmic staining, with additional labeling of the nucleolus (arrow). Within the nucleoplasm, the antibody decorates nuclear speckles. Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 2. The full-length (150 kDa) form of hADAR1 localizes to the cytoplasm and the short (110 kDa) form localizes to the nucleolus. (A) Schematic representation of differently tagged hADAR1 constructs. In-frame methionines present in hADAR1 cDNA are shown (M296, M337). The NLS recently described by Eckmann et al. (Eckmann et al., 2001) is marked by an asterisk (*). (B) HeLa cells were transiently transfected with the indicated hADAR1 constructs and assayed for ADAR1 localization by either direct detection of GFP or indirect immunofluorescence using anti-FLAG and anti-His antibodies. GFP- and FLAG-tagged ADAR1 localize to the cytoplasm. The anti-His antibody stains both the cytoplasm and the nucleolus. In addition, the anti-His antibody labels discrete aggregates in the nucleoplasm (arrow). (C) Western-blot analysis of total proteins from COS7 cells transfected with the indicated plasmids. Total cell extracts after 36 hours of expression were prepared and fractionated by SDS-PAGE. Western blotting was then performed with an anti-GFP antibody (lanes 1, 2), an anti-FLAG antibody (lanes 3, 4) and an anti-His antibody (lanes 5-7). The western-blot signal observed with mobility just below that of the ADAR1 band with the anti-FLAG antibody is a nonspecific signal also present in non-transfected cells (Fig. 2C, lanes 3,4). Both antibodies recognize the full-length (150 kDa) hADAR1 but the short (110 kDa) form of the protein is only specifically detected with anti-His antibody. Molecular weight markers are shown on the left.

View larger version (45K): [in a new window]   Fig. 3. hADAR1 shuttles between the cytoplasm and the nucleus and localizes to the nucleolus with no requirement for the N-terminal domain. N-terminal and C-terminal deletions of hADAR1 were fused to GFP. The most important domains of hADAR1 are indicated as follow: ZBDs, Z-DNA-binding domains (light gray); dsRBDs, double-stranded RNA binding domains (black boxes); deaminase domain (dark gray box). Numbers denote amino-acid positions relative to the N-terminus of hADAR1. HeLa cells were transfected with the indicated constructs and, approximately 18 hours after transfection, the cells were incubated with or without 50 nM LMB for 3 hours, fixed and observed directly with the fluorescence microscope. Notice that the cytoplasmic chimera resulting from the C-terminal deletion (C) accumulates in the nucleus with nucleolar exclusion after LMB treatment (D), whereas the full-length fusion protein (A) is concentrated in the nucleolus after LMB treatment (B). HeLa cells transfected with GFP-ADAR1C-Term were fused with murine 3T3 cells to form heterokaryons. These cells were treated with a protein synthesis inhibitor (emetine, 20 µg ml-1) for 3 hours before fusion. After fusion, the cells were kept in culture for 3 hours in the presence of emetine. Heterokaryons were fixed and labeled with monoclonal antibody 4F4 directed against hnRNP C protein, which does not shuttle. Like hnRNP C (F), the N-terminal deletion of hADAR1 remains restricted to the transfected HeLa cell nucleus (E). The dashed lines in E and F indicate the contour of the murine nucleus in the heterokaryon. Notice that the N-terminal deletion of ADAR1 localizes exclusively to nucleoli (E). Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 4. Subcellular distribution of endogenous ADAR2. Schematic diagram comparing hADAR1 and hADAR2. The numbers indicated at the C-terminus represent size of the protein in amino acids. The most important domains of hADAR1 are indicated as follow: ZBDs, Z-DNA-binding domains (light gray); dsRBDs, double-stranded RNA binding domains (black boxes); deaminase domain (dark gray box). (A) HeLa cells were immunolabeled with anti-ADAR2 antibody. The labeling pattern is diffuse throughout the nucleoplasm, with concentration in nucleoli. The relative concentration of ADAR2 staining in the nucleolus varies from cell to cell. (B) A western-blot analysis of total (T) and nuclear (N) HeLa cell extracts. Cell extracts were prepared and fractionated by SDS-PAGE. Western blotting was then performed with anti-ADAR2 antibody (Ab 70). Extracts of HeLa cells transiently transfected with FlisADAR2 are shown in lane 3. Molecular weight markers are shown on the left. (C,D) Cryosections of neurosecretory neurons from the hypothalamic supraoptic nucleus were double-labeled with antibodies directed against fibrillarin (C) and ADAR2 (D). ADAR2 is predominantly detected in nucleoli, which can be readily identified by the fibrillarin staining. Bar, 10 µm.

View larger version (156K): [in a new window]   Fig. 5. Electron microscopy reveals that ADAR2 localizes in a novel subnucleolar compartment. Ultrathin sections from rat brain neurons were immunogold labeled with antibodies directed against ADAR2 and fibrillarin. The ADAR2-specific gold particles decorate the periphery of the fibrillar component (f) and are largely excluded from the fibrillar centers (fc) and the granular component (g). By contrast, the anti-fibrillarin antibody produces an intense labeling of the dense fibrillar component. Bar, 300 nm.

View larger version (133K): [in a new window]   Fig. 6. hADAR1 and hADAR2 co-localize within the nucleolus. HeLa cells were transfected with GFP-ADAR2, incubated in the absence (A) or in the presence (B) of actinomycin D (0.07 µg ml-1) for 1 hour and double-labeled with a monoclonal antibody against fibrillarin. The merged images show clearly that GFP-ADAR2 (green) and fibrillarin (red) do not co-localize. (C) HeLa cells were co-transfected with GFP-ADAR2 and Flis-ADAR1C-Term (Fig. 2A). Approximately 16 hours after transfection, cells were fixed, immunostained with anti-FLAG antibody (red) and analysed by confocal microscopy. GFP-ADAR2 and Flis-ADAR1 co-localize in the nucleolus of co-transfected cells, as can be seen in the merged image (yellow). Images from the same microscopic fields are shown side by side. The dashed lines indicate the contour of the HeLa cell nucleus. Bar, 10 µm.

View larger version (43K): [in a new window]   Fig. 7. hADAR2 does not shuttle and an N-terminal domain is responsible for its nuclear localization. (A) Schematic representation of the GFP-tagged full-length hADAR2 and the GFP-ADAR2 deletions constructed to study putative localization signals in hADAR2. Numbers denote amino acid positions relative to the N-terminus of hADAR2. Asterisks indicate putative NLSs identified by sequence analysis, and the respective amino acid sequences are shown. (B) HeLa cells were transiently transfected with the indicated GFP-ADAR2 constructs. Approximately 16 hours after transfection, cells were fixed and directly observed with the fluorescence microscope. (C) HeLa cells were transfected with GFP-ADAR2 and fused with murine NIH 3T3 cells. The heterokaryons were incubated in the presence of emetine. Immunostaining with anti-hnRNP C was used as control. Both GFP-ADAR2 and hnRNP C molecules remain restricted to the transfected HeLa nucleus. The dashed lines indicate the contour of the murine nucleus in the heterokaryon. Incubation of HeLa cells expressing GFP-ADAR2 at 4°C in the presence of emetine shows that the protein does not leak to the cytoplasm. Bar, 10 µm.

View larger version (36K): [in a new window]   Fig. 8. hADAT1 is a nucleocytoplasmic shuttling protein that is excluded from the nucleolus. Schematic representation of the homology between hADAR1, hADAR2 and hADAT1. The three enzymes contain a deaminase domain and only hADAT1 does not contain dsRNA-binding domains. The most important domains of hADAR1 are indicated as follow: ZBDs, Z-DNA-binding domains (light gray); dsRBDs, double-stranded RNA binding domains (black boxes); deaminase domain (dark gray box). The size of the proteins are indicated at their C-termini as the number of amino acids. HeLa cells were transfected with GFP-ADAT1 and fused with murine NIH 3T3 cells. The heterokaryons were incubated in the presence of emetine. Immunostaining with anti-hnRNP C was used as control. In contrast to hnRNP C, which remains restricted to the transfected HeLa nucleus, GFP-ADAT1 migrates to the murine nuclei. The dashed lines indicate the contour of the murine nuclei in the heterokaryon. Incubation of HeLa cells expressing GFP-ADAT1 at 4°C in the presence of emetine shows that the protein does not leak to the cytoplasm. Bar, 10 µm.

View larger version (66K): [in a new window]   Fig. 9. Endogenous hADAR1 and hADAR2 are excluded from the nucleolus of cells expressing an editing substrate. HeLa cells were transiently transfected with a plasmid containing either the editing-competent murine GluR-B gene portion (Minigene B13) or a control gene portion from the Friends virus genome (C-RNA). Endogenous localization of editing enzymes was monitored by indirect immunofluorescence microscopy with antibodies directed against ADAR2 (b,e,n) and ADAR1 (Ab 668) (h). Alternatively, cells were transfected with a plasmid encoding GFP-ADAR2 (l). GluR-B- and C-RNA-transcribing cells were visualized by fluorescent in situ hybridization with a GluR-B (a,d,g,j,p) or C-RNA (m) probe labeled with digoxigenin. The hybridization sites were detected using a cy3 anti-digoxigenin secondary antibody. The GluR-B and C-RNA transcripts were detected 40 hours after transfection. The GluR-B probe produces mainly a nucleoplasmic staining pattern, with nucleolar exclusion (a,d,g,j,p). In addition, transcripts tend to accumulate in discrete nucleoplasmic regions (d,g,j,p). In GluR-B-transcribing cells, ADAR2 and ADAR1 become completely excluded from the nucleolus (b,e,h,l). For comparison, cells that do not express GluR-B are shown in b (left side), h (inset) and l (inset). (e,h,l) Recruitment of ADAR1 and ADAR2 to the nucleoplasmic regions where GluR-B transcripts accumulate. In contrast to cells expressing GluR-B, ADAR2 persists concentrated in the nucleolus of cells expressing C-RNA (m-o). Furthermore, in contrast to ADAR1 and ADAR2, the nucleolar protein B23 remains in the nucleolus of cells expressing GluR-B transcripts (p-r). (c,f,i,k,o,r) A superimposition of the corresponding double-labeled images. Bar, 10 µm.

View larger version (42K): [in a new window]   Fig. 10. FRAP and FLIP analysis of GFP-ADAR2 and GFP-ADAR1C-Term. (A) HeLa cells expressing either GFP-ADAR2 or GFP-ADAR1C-Term were bleached in a selected nucleolus for 0.5 seconds. Images were taken before bleaching and at the indicated time points after the end of the bleach pulse. (B) HeLa cells expressing either GFP-ADAR2 or GFP-ADAR1C-Term were repeatedly bleached in a selected nucleolus at intervals of 4.5 seconds. Images were taken before bleaching and at the indicated time points after the end of the first bleach pulse. The bleached nucleolus is indicated by an arrow in the pre-bleached panels. The dashed lines indicate the contour of the HeLa cell nucleus. Corresponding quantitative data of fluorescence-recovery kinetics are plotted on the right-hand side of each set of images. For FRAP experiments (A), the fluorescence intensity in the bleached region was measured and expressed as a relative fluorescence recovery (normalized fluorescence; see Materials and Methods). For FLIP experiments (B), the fluorescence intensity was measured in an adjacent nucleolus (marked by a white circle) and normalized as in FRAP experiments. Bar, 10 µm.

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