42 The Journal of Cell Biology | Volume 156, Number 1, 2002 neurons and fibroblasts may share a similar mechanism for sorting of -actin mRNA. We have previously reported a cis-acting element, the zip- code, which is necessary and sufficient for asymmetric segre- gation of -actin mRNA in fibroblasts. Deletion or muta- tion of the zipcode delocalized a reporter mRNA, and antisense treatment of the zipcode affected the regional syn- thesis of -actin protein and, as a consequence, the cell mo- tility (Kislauskis et al., 1994, 1997; Shestakova et al., 2001; Zhang et al., 2001). A cytoplasmic trans-acting factor, zip- code binding protein(ZBP)1, has been characterized (Ross et al., 1997); it bound to the zipcode of -actin mRNA, but did not bind to a mutated zipcode incapable of asymmetri- cally localizing a reporter. Recently, the Xenopus homologue of ZBP1 has been identified by virtue of its binding to a lo- calization element in Vg1 mRNA, an RNA that becomes lo- calized to the vegetal pole of oocytes (Deshler et al., 1998; Havin et al., 1998). This implies that a common machinery may exist for targeting different mRNAs in diverse cell types. It is likely that ZBP1 is a member of the locasome, a com- plex of proteins specialized for localization (Bertrand et al., 1998). Because -actin mRNA is also localized in neurons, we searched for the complex in brain extracts. In this work we report a second protein that binds to the zipcode and is highly enriched in brain. We show that ZBP2 is a homo- logue of human hnRNP protein, KSRP, that regulates premRNA splicing (Min et al., 1997). Interestingly, ZBP2, like KSRP, is predominately a nuclear protein. The in vitro and in vivo data suggest that ZBP2 also has a small fraction present in the cytoplasm and may spend a short time in the cytoplasm, and in this way may contribute to the subcellular localization of -actin mRNA. Results Identification of zipcode binding proteins To identify proteins that bound to the zipcode of -actin mRNA, RNA mobility shift and UV crosslinking approaches were used (Ross et al., 1997). Radiolabeled zipcode tran- scripts were used for RNA mobility shift assays. Three dis- tinct RNA–protein complexes were formed when the labeled zipcode probe was incubated with brain or fibroblast extracts (Fig. 1 A, lanes 1–4; complexes are indicated by arrows). The intensity of shifted complexes was proportional to the amount of brain extract used (lanes 1–3). The fast-migrating complex (F) was seen in both brain and fibroblast extracts (lanes 3 and 4, respectively). However, the two slower mi- grating complexes (M and S) were more evident in brain than in fibroblast extracts (lanes 3 and 4). The specificity of the complexes was determined by competition assays in which 100-fold excess of unlabeled zipcode competed with the for- mation of the complexes (lane 5), whereas 100-fold excess of unlabeled RNA transcribed from pGEM 3Z vector did not interfere with the complex formation (unpublished data). Because a mutated zipcode failed to localize the reporter mRNA (Ross et al., 1997), we reasoned that the mutant zip- code would not form an RNA–protein complex. To test this, we generated radiolabled mutant zipcode in which the two ACACCC motifs were replaced by GTGTGT, and we performed RNA mobility shift assays with brain extracts (Fig. 1 B). In contrast to the wild-type zipcode (lane 1), the mutant zipcode failed to form RNA–protein complexes (Fig. 1 B, lane 2). This indicated that proteins in the brain extract did not bind to the mutant zipcode, and demon- strated that a mutation that impairs mRNA localization can be correlated with impaired binding of the RNA–binding protein. To distinguish the protein components that bind to the zipcode, we used UV to crosslink RNA–protein com- plexes formed by mixing the radiolabeled zipcode with brain extracts, and analyzed the complexes by SDS-PAGE. All three RNA–protein complexes were not detectable without crosslinking (Fig. 1 C, no UV-X). When the RNA–protein complexes were crosslinked and stabilized, specific bands were visualized (UV-X). Complex F contained a crosslinked protein with an estimated molecular mass of 68 kD, the same molecular mass as ZBP1 (Ross et al., 1997). Com- Figure 1. Characterization of proteins binding to the zipcode in chicken embryo brain and fibroblast extracts. (A). Complexes formed from brain and fibroblasts. [32P]-labeled transcripts encoding the 54 nts zipcode of the 3 UTR of chicken -actin mRNA were incubated with aliquots of chicken embryo brain and fibroblast protein extracts, followed by incubation with RNase T1 (50 U/ml) and heparin (5 mg/ml). RNA–protein complexes were resolved in 4% polyacrylamide native gels that were then dried and exposed to Kodak X-Ray film. (Lanes 1, 2, and 3) [32P]-labeled transcript incubated with 1-, 2-, and 5- l brain extracts, respectively. (Lane 4) [32P]-labeled transcript incubated with 5 l fibroblast extract. (Lane 5) [32P]-labeled transcript incubated with 5- l brain extract in the presence of 100 excess molar amounts of unlabeled 3 UTR of actin mRNA transcripts. The arrows point to the slow (S), medium (M), and fast-migrating (F) RNA–protein complexes. (B) Specificity of the complexes for the zipcode. (Lane 1) Zipcode incubated with brain extract. (Lane 2) Mutant zipcode incubated with brain extract. (C) UV crosslinking assay. [32P]-labeled transcripts were incubated with brain extract, treated with RNase T1, and exposed to UV light for 3 min at a distance of 1 cm. Each of the RNA–protein complexes (F, M, and S) was identified and excised from a 4% polyacrylamide native gel, soaked in SDS buffer, and resolved in 10% SDS-PAGE. (Left three lanes) Excised RNA–protein complexes F, M, and S that were not UV crosslinked. (Right three lanes) Excised RNA–protein complexes F, M, and S that were crosslinked. The arrows indicate the protein mobilities with labeled RNA bound.