48 The Journal of Cell Biology | Volume 156, Number 1, 2002 Figure 9. A ZBP2 dominant negative construct suppresses -actin mRNA localization. (Top) The percentage of transfected CEFs wth localized -actin mRNA. CEFs were transfected for 6 h followed by fluorescence in situ hybridization. Transfected cells were scored for -actin mRNA localization. GFP, vector pEGFPC1; GFP-CD, GFP fused with central domain; GFP-KSRP, GFP-human KSRP; Mock, no plasmid. The bars show percentage of transfected cells with localized -actin mRNA in each experiment. At least 50 transfected cells were counted in each coverslip, two experiments each. (Middle) The -actin mRNA signal in neuronal processes. Experiments were parallel to A except that the COOH terminus of ZBP2 (GFP-CT) was used (Fig. 8, construct B). -actin mRNA signal was measured and then converted into fluorescence units. (Bottom) The percentage of cotransfected CEFs with local- ized -actin mRNA. CEFs were transfected either with single construct (wild-type ZBP2 or the dominant negative central domain construct) or both constructs of different ratios (dominant negative vs. wild-type 1:1 or 1:4) for 6 h. After FISH, transfected cells were scored as in Fig. 9 A. in this complex have homology to the transcription factor, FBP1 (Duncan et al., 1994), a single-stranded DNA bind- ing protein, ssDBF (Smidt et al., 1995) also homologous to Drosophila Squid, known to localize ftz (Lall et al., 1999), and an A/B hnRNP associated with mRNA editing (Lau et al., 1997). All of these are likewise nuclear in location and function, and some may shuttle between the nucleus and cy- toplasm and could play a concerted role in vetting the RNA before, during, or after export. The amino acid sequence predicted from ZBP2 cDNA demonstrates that ZBP2 is a chicken homologue of human KSRP, a regulatory splicing factor (Min et al., 1997). Like ZBP1 (Ross et al., 1997), KSRP/ZBP2 contains four KH domains, a glutamine-rich COOH-terminal domain, and a proline, glycine-rich NH2-terminal domain that may pro- vide flexibility for the protein to fold properly. In addition, ZBP2 is particularly abundant in cell nuclei where it could also be involved in splicing of many pre-mRNAs, possibly including -actin, as well as their localization. Our results provide evidence that ZBP2 interacts with the zipcode in vitro and participates in -actin mRNA localiza- tion in vivo. First, the recognition of the zipcode by ZBP2 is specific: it does not bind to nonzipcode RNA. This was dem- onstrated using RNA competition and immunodepletion as- says wherein the formation of the RNA–protein complexes was abolished. That the mutant zipcode failed to bind ZBP2 in vitro is consistent with the previous study showing that mutated zipcode delocalized -actin mRNA in vivo (Ross et al., 1997). Second, we verified the physical association of ZBP2 with the zipcode using UV to covalently crosslink it to the zipcode, and by immunoprecipitation where the -actin mRNA copurified with it in brain extracts. Third, immuno- cytochemistry and in situ hybridization showed ZBP2 present infrequently in the cytoplasm, where it colocalized with -actin mRNA at the leading edge of fibroblasts and growth cones of developing neurons. Moreover, EGFP–ZBP2 trans- fected into fibroblasts, although also infrequently cytoplasmic, showed a similar colocalization pattern to the endogenous ZBP2 and -actin mRNA, suggesting that the movement of ZBP2 toward the cell’s leading edge was associated with local- ization of the mRNA. Importantly, the expression of a trun- cated construct of ZBP2 containing the central KH domains and the nuclear localizer 47 aa interfered with endogenous -actin mRNA localization. ZBP2, a predominantly nuclear protein, is shown here to play a role in cytoplasmic transport of -actin mRNA by bind- ing to the localization signal. Of interest, ZBP2 has similarities to ZBP1, a predominantly cytoplasmic protein, which also is involved in -actin mRNA localization in fibroblasts and neu- rons (Zhang et al., 2001). However, unlike ZBP2, ZBP1 has a nuclear export signal (Ross et al., 1997), which may explain its predominate cytoplasmic localization. Although both ZBP1 and ZBP2 are in different cellular compartments, they share many common features. Both ZBP1 and ZBP2 recognize the wild-type zipcode, but not the mutant zipcode in vitro, and colocalize with -actin mRNA in vivo (Ross et al., 1997; Zhang et al., 2001). Therefore, the two hnRNP proteins, one cytoplas- mic and the other nuclear, are involved in localization of the same mRNA. We propose that the process for -actin mRNA localization may initiate in nuclei where ZBP2 may interact