The third dsRBD is important for nuclear import of hsADAR1. To determine the importance of the third dsRBD for nuclear import of hsADAR1, this domain was removed from the full-length protein. To avoid nuclear export of the overexpressed proteins cells were treated with LMB before fixation and staining. myc-tagged, full-length hsADAR accumulates in the nucleus (hsFL+LMB), whereas deletion of the third dsRBD in this protein leads to cytoplasmic accumulation (hsFLΔds3). FITC, Localization of myc-tagged constructs seen after antibody staining. DAPI, Localization of nuclei as seen in the DAPI channel. NOM, Whole cell images were taken by Nomarski optics. Scale bar, 20 μm.

RNA binding is not required for NLS activity of hsNLS35, the third dsRBD of hsADAR1. To determine whether hsNLS35 acts via binding to an RNA that is imported into the nucleus, transcription was inhibited with the use of AMD. The third dsRBD has NLS activity (hsNLS35) even in the absence of transcription (hsNLS35+AMD). A single (hsNLS35-H754A) or double-point mutation (H754A+F758A) in this domain that abolish RNA binding has no effect on NLS activity. myc-tagged proteins are shown in the FITC channel (FITC), and nuclei were stained with DAPI (DAPI). Whole cells were imaged by differential interference contrast (NOM). Scale bar, 20 μm.

xlNLS12 is necessary and sufficient for nuclear import in HeLa cells. HeLa cells grown on coverslips were transiently transfected with the PK-myc constructs indicated. After fixation cells were permeabilized and stained for the presence of myc-tagged protein with the use of mAb 9E10 and a secondary FITC-labeled antibody (FITC). xlNLS12 displays NLS activity, whereas the R508G point mutation in this construct does not. myc-tagged full-length xlADAR1.1 protein accumulates in the nucleus (xlFL). The single-point mutation R508G in xlADAR1.1 abolishes nuclear accumulation (xlFL R508G). Nuclei were stained with DAPI and images of whole cells were taken by differential interference contrast (NOM). Scale bar, 20 μm.

The third dsRBD of hsADAR1 has NLS activity. Various fragments of the hsADAR1 protein were fused to a PK-myc reporter construct, transfected into HeLa cells, and tested for NLS activity. HsNLS2, the region corresponding to the NLS found in xlADAR1 displays no NLS activity. Similarly, a large amino-terminal fragment of hsADAR1, harboring two additional putative NLSs, is also inactive (hsNLS9). In contrast, the third dsRBD of hsADAR1 displays NLS activity (hsNLS35). Deletion into this dsRBD from either the C-terminal end (hsNLS29) or the N-terminal end (Eckmann, Neunteufl, Pfaffstetter, and Jantsch, unpublished results) completely abolishes NLS activity. FITC, Localization of myc-tagged protein as seen after antibody staining in the FITC channel. DAPI, Nuclei counterstained with DAPI. Whole cells were imaged by differential interference contrast (NOM). Scale bar, 20 μm.

xlNLS12 is both necessary and sufficient for nuclear import in Xenopus oocytes. Oocytes were injected with RNAs encoding myc-tagged xlNLS-PK reporter constructs, myc-tagged full-length (xlFL) or mutated xlADAR1 (xlFL R508G). Extracts of hand-enucleated oocytes corresponding to one cytoplasm (CP) and five GVs were loaded on a 7% SDS-PAGE gel and blotted, and myc-tagged proteins were detected with mAb 9E10. (A) Constructs covering the two putative amino-terminal NLSs (xlNLS1 and xlNLS2) or the entire amino-terminal region of xlADAR1 (xlNLS3) show no nuclear accumulation. In contrast, xlNLS12 shows clear nuclear accumulation, whereas the point mutation xlNLS12 R508G does not. Uninjected oocytes show no signal (CON). (B) Full-length xlADAR (xlFL) accumulates in the nucleus, whereas the point mutation R508G in this protein (xlFL R508G) abolishes nuclear accumulation. To fit the intensity of bands the lane containing the xlFL extract was exposed longer.

xlADAR1 is constitutively nuclear in HeLa and Xenopus cells. (A) To determine whether nuclear accumulation of xlADAR1 is transcription dependent, HeLa cells were transfected with a myc-tagged, full-length xlADAR1 protein (xlFL). The ectopically expressed protein is constitutively nuclear in both the absence (CON) and presence of the transcriptional inhibitor AMD (AMD) as can be seen in the FITC channel. In contrast, staining with an antiserum specific for the human ADAR1 protein indicates that this protein is translocated to the cytoplasm upon AMD treatment (endog. TRITC). Note that the localization of endogenous hsADAR1 is not affected by overexpression of the Xenopus ADAR1 protein, suggesting different import mechanisms for those two proteins. (B) Also in Xenopus cells xlADAR1 is constitutively nuclear. Endogenous xlADAR1 protein was visualized in the tetramethylrhodamine (TRITC) channel with a rabbit polyclonal antiserum specific for Xenopus ADAR1 (endog. TRITC) in the absence (CON) or presence of AMD (AMD). DAPI, Nuclei were visualized by DAPI staining. NOM, Whole cells were imaged by Nomarski optics. Scale bar, 20 μm.

Schematic representation of Xenopus and human ADAR1 proteins and fragments tested for NLS activity. Only most important fragments are shown. Fragments that displayed NLS activity are highlighted by bold lines. Important elements found in those proteins are indicated as follows: ZBDs (hatched boxes), dsRBDs (dark gray boxes), deaminase domain (striped box). Previously identified putative NLSs are indicated by open arrowheads. The actual active NLSs are marked by inverted black triangles. (A) Schematic drawing of Xenopus ADAR1.1 and fragments tested from this protein. The sequence of the minimal xlNLS12 fragment is displayed on top. The single-point mutation R508G that abolishes NLS activity both in xlNLS12 and in the full-length protein is indicated. (B) Schematic drawing of human ADAR1. The sequence of the minimal active hsNLS35 fragment is shown at the bottom. The dsRBD consensus sequence is highlighted in bold face italics. Deletions into this region that abolish NLS activity are boxed in gray. Two mutations, H754A and F758A, that abolish RNA binding but do not affect NLS activity as well as a C773A mutation that changes a Cys found conserved in some mammalian ADAR1 proteins are indicated.

Nuclear accumulation of hsADAR1 is regulated. (A) Nuclear accumulation of transiently transfected hsADAR1 is concentration dependent and can be stimulated by LMB. Strong transient expression of myc-tagged hsADAR1 leads to cytoplasmic accumulation of this protein (hsFL). In cells selected for low levels of expression after G418 treatment, the protein can efficiently move to the nucleus (hsFL+G418). LMB treatment leads to nuclear accumulation of full-length hsADAR1 (hsFL+LMB), suggesting that cytoplasmic accumulation of overexpressed hsADAR1 is caused by increased nuclear export rather than reduced nuclear import. As can be seen cytoplasmic staining can still be observed in most of the cells. In only 20% of cells is complete nuclear accumulation of the transfected protein observed. Removal of LMB leads to redistribution of ectopically expressed hsADAR into the cytoplasm, indicating that the observed block of nuclear export by LMB treatment is reversible (hsFL+LMB+wash). (B) Nuclear accumulation of endogenous hsADAR1 is transcription dependent but can be stimulated by LMB. Staining with an antibody directed against endogenous hsADAR1 indicates that the protein is predominantly but not exclusively nuclear in normally growing cells (endog.). Inhibition of transcription by AMD treatment relocates the protein from the nucleus to the cytoplasm, indicating that ongoing transcription is required for nuclear accumulation or retention of the endogenous protein (endog.+AMD). LMB treatment leads to nuclear accumulation of endogenous hsADAR1 protein (endog.+LMB). The cytoplasmic staining seen in untreated cells is reduced, whereas nuclear staining is increased after LMB treatment. FITC, Localization of myc-tagged transfected (A) or endogenous (B) protein seen after antibody staining in the FITC channel. DAPI, Staining of nuclei with DAPI. NOM, Images of whole cells taken by Nomarski optics. Bar, 20 μm.