A nuclear protein affecting -actin mRNA localization | Gu et al. 47 Figure 8. Nuclear versus cytoplasmic distribution of ZBP2 in fibroblasts and neurons. The gene structure of ZBP2 is demonstrated in A. Red bar, 47-aa insert; blue bars, KH domains; gray bars, COOH-terminal repeat of AWEEYYK motif. The various constructs of ZBP2; the full-length with or without the 47-aa insert (F and G), the 47-aa insert (E), the four KH domains (D), the central domain containing the four KH domains, and the 47-aa insert (C), or the COOH terminus of the protein (B) were fused to GFP and the cellular distribution characterized. The constructs are detailed in the left panels with cellular distributions in the right panels. The percent of cells with cytoplasmic signal was characterized for each construct in the number under each construct. Bars, 10 m. and hence act as a dominant negative for -actin mRNA lo- calization. The EGFP signal in fibroblasts transfected with the truncate was mainly present in the nucleus. Localization of the -actin mRNA was visualized by fluorescence in situ hybridization. The percentage of cells with localized -actin mRNA decreased roughly by half in the transfected CEFs (Fig. 9, top) in contrast to the cells transfected with EGFP or EGFP-KSRP as a control (the human ZBP2 homologue). The intensity of cytoplasmic signal of -actin mRNA did not change significantly, indicating that the nuclear export of -actin mRNA was not inhibited by the dominant nega- tive construct (unpublished data). In transfected developing neurons, this truncation also caused a 35% decrease of -actin mRNA signal in processes or growth cones compared with the control experiment (Fig. 9, middle). This suggested that the overexpression of the truncated, but still nuclear, ZBP2 affected the -actin mRNA localization. To demon- strate that the dominant negative effect of ZBP2 truncate is specific, we performed cotransfections of both the full- length ZBP2 and central domain into CEFs. The delocaliza- tion of -actin mRNA in transfected cells was partially res- cued by using the wild-type ZBP2 (Fig. 9, bottom). Discussion We have purified and identified five proteins from embry- onic brain extracts that bind to the zipcode sequence respon- sible for localizing -actin mRNA in fibroblasts and neu- rons. One of these has been previously identified from cultured fibroblast extracts as ZBP1 (Ross et al., 1997). In this work we have characterized ZBP2, a ZBP highly en- riched in chicken brain tissue. The binding specificity of this protein to the zipcode, its intracellular colocalization with -actin mRNA, and the dominant negative effect of a trunca- tion of this protein suggests a role in localization of -actin mRNA. Consistent with its homology to the human splicing factor, KSRP, the protein is predominantly nuclear in its in- tracellular distribution and the possibility exists that it exerts its effect there with subsequent consequences for the cyto- plasmic compartmentalization. The other proteins identified