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.