46 The Journal of Cell Biology | Volume 156, Number 1, 2002 Figure 6. Distribution of -actin mRNA and ZBP2 in CEFs and neurons. CEFs (A) and neurons (B) were cultured and fixed as described in Materials and methods. The cells were in situ hybridized to -actin mRNA (Cy3, red) and by immunofluorescence to ZBP2 (FITC, green). Overlap is indicated in the right of A and in B. Image in B is a superimposed single, deconvolved optical sections. Arrowheads show the colocalization of ZBP2 and -actin mRNA in neuronal processes and growth cone. Bars, 10 m. the COOH-terminal repeats (gray) (Fig. 8 A). Different do- mains of ZBP2 control the nuclear or cytoplasmic compart- mentalization of the EGFP fused to it (numbers indicate the percentage of cells with cytoplasmic fluorescence in fibro- blasts). The COOH-terminal (Fig. 8 B) domain and four KH domains (Fig. 8 D) were distributed throughout the cell, whereas when the 47 aa were added to the 4-KH domain, which we call the central domain, the localization was almost entirely nuclear (Fig. 8 C). This suggested that the 47 aa de- termined nuclear distribution. This was confirmed by fusing the 47-aa segment to EGFP, which then became exclusively nuclear (Fig. 8 E). Inspection of the 47-aa segment revealed several arginine residues suggestive of a nuclear localization signal (NLS). When the full-length ZBP2 was fused to GFP, with or without the 47-aa segment (Fig. 8, F and G), the nu- clear fluorescence was decreased but not eliminated in the construct without the 47 aa, and the cytoplasmic fluorescence increased, indicating that weak NLSs most likely existed else- where in the protein. The distribution in fibroblasts for these last two constructs was quantitated in neurons, and yielded the same percentages of cells with cytoplasmic fluorescence. Unlike ZBP2, human KSRP was entirely nuclear when fused to GFP and expressed in CEF (unpublished data). Overexpression of ZBP2 central domain partially disrupts -actin mRNA localization in CEFs and neurons A truncate of the ZBP2, the central domain, which con- tained 400 aa including the 47 aa with four KH domains (Fig. 8 C), was transfected into CEFs. We postulated that the KH domains would compete for RNA binding with the full-length ZBP2, analogous to ZBP1 (unpublished data), Figure 7. -actin mRNA was coprecipitated with ZBP2. ZBP2 anti- bodies were used to immunoprecipitate a CEF extract. Primers to -actin mRNA were used in an RT-PCK assay to detect the presence of -actin mRNA in the pellets. (Lane 1) Negative PCR control no primers. (Lane 2) Preimmune rat serum. (Lane 3) ZBP2 antiserum. (Lane 4) Purified rat normal IgG. (Lane 5) affinity-purified ZBP2 IgG. (Lane 6) Positive PCR control using full-length cDNA of -actin mRNA as template. M is the DNA molecular marker. The arrow denotes the PCR amplified DNA fragment of the 3 UTR of -actin mRNA.