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.

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