with the zipcode, perhaps with other proteins, such as ssDBF and FBP. Upon nuclear export of the RNA, ZBP1 may take over the process of cytoplasmic localization. The fact that the proteins form different complexes with the zipcode suggests that they bind sequentially rather than simultaneously. Increasing evidence demonstrates that specific interactions between RNA localization elements and cellular factors play an essential role in cytoplasmic sorting of mRNAs to their destinations. Strikingly, although mRNA localization is a cytoplasmic event, proteins that shuttle between the nucleus and cytoplasm participate in this pathway. A number of other nuclear proteins participating in cytoplasmic mRNA localization have been documented in recent studies (Hoek et al., 1998; Cote et al., 1999; Lall et al., 1999; Long et al., 2001). VgRBP60, a hnRNP I type protein in Xenopus, binds to the VM1 localization motif of Vg1 mRNA in vitro and colocalizes with Vg1 mRNA in vivo (Cote et al., 1999). Lall et al. (1999) reported that sqd, a Drosophila hnRNP protein is required for ftz mRNA localization in embryos. In yeast, an exclusively nuclear protein (does not shuttle), Loc1p, binds to the 3 UTR zipcode of ASH 1 mRNA and is re- quired for its efficient cytoplasmic localization to the bud tip (Long et al., 2001). Therefore, our finding that ZBP2, an hnRNP protein with known nuclear location, was involved in cytoplasmic localization of -actin mRNA was consistent with hnRNPs being part of a common localization mecha- nism. It is possible that hnRNPs including ZBP2/KSRP are required for packaging the RNA in nucleus in a way that marks it for a localization pathway in the cytoplasm. The fact that ZBP2 is only found in the cytoplasm of 5–10% of the cells strongly suggests that it rapidly shuttles, spending a short time there. This is the opposite of ZBP1, which spends only a short time in the nucleus (unpublished data) and is predominantly cytoplasmic. The 47-aa segment has a strong effect on ZBP2 nuclear retention, just as the nu- clear export signal in ZBP1 has a strong effect on its cytoplas- mic presence (unpublished data). When removed from ZBP2, the 47-aa segment increases the presence in the cytoplasm. KSRP, which does not have this 47-aa segment, also does not appear in the cytoplasm, at appreciable levels. Possibly KSRP is an alternatively spliced variant of ZBP2, but we have not found evidence for this in chicken, which does not appear to have KSRP. Like KSRP, ZBP2 is a member of a KH domain– containing family of neuronally expressed proteins which in- clude KSRP, ZBP1, FMRP (Fridell et al., 1996), and NOVA (Polydorides et al., 2000), all involved in some aspects of the nuclear regulation of RNA. Microsequencing of the other proteins copurified with ZBP2/KSRP by RNA affinity selection showed that the 70- kD protein is also a KH domain containing protein, a ho- mologue of FBP, a human transcription factor, that binds to single-stranded DNA and activates the transcription of the c-myc gene (Duncan et al., 1994). It is possible that FBP was copurified with ZBP2 because it has been shown that FBP associates with KSRP in human nuclear extracts (Min et al., 1997). However, this does not eliminate the possibility that FBP may have a function in cytoplasmic mRNA distribu- tion. The 45-kD protein, which was copurified with ZBP2, is ssDBF, a nuclear hnRNP A/B type protein that binds to the regulatory site of apo VLDL II gene (Smidt et al., 1995), A nuclear protein affecting -actin mRNA localization | Gu et al. 49 and a human homologue of ssBDF is involved in Apo mRNA editing (Lau et al., 1997). ssDBF shares 74% identity with MBP mRNA binding protein (Hoek et al., 1998), and 41% identity with sqd, the ftz binding protein in Drosophila (Lall et al., 1999), both of which are also hnRNA A/B proteins. Whereas both MBP mRNA binding protein and sqd appear to perform an essential role for localization of their respective mRNAs, ssDBF involvement in cytoplasmic -actin mRNA segregation remains to be determined. These proteins could be part of a complex that we have termed the locasome (Bertrand et al., 1998). This structure most likely contains proteins unique to both the nucleus and the cytoplasm, in the first case marking the RNA for local- ization, and in the second case directing the peripheral loca- tion to the leading edge of the fibroblast or the growth cone of the developing neuron. Materials and methods Preparation of chicken brain and fibroblast extracts Brain cytoplasmic extract was prepared from 12-d-old chick embryos. Whole brains were removed and washed three times with buffer A (20 mM Tris Cl, 3 mM MgCl2, 40 mM KCl and 1 mM DTT, 0.7 g/ml leupeptin, 1 g/ml aproti- nin, 0.7 g/ml pepstantin and 1 mM PMSF). 1 ml of buffer A was added per gram of wet tissue, and the mixture was homogenized in a Teflon glass ho- mogenizer. Homogenates were centrifuged for 10 min at 5,000 g, and the su- pernatant was centrifuged for 2 h at 28,000 rpm in a Beckman SW-40.1 rotor. The high-speed supernatant was either used immediately or stored in aliquots at –70 C. The protein concentration of the brain extract was 10–20 g/ l. To prepare CEF extract, fibroblasts were isolated from breast muscle tissue of 11-d-old chick embryos. Cells were grown to 95% confluency (Kislauskis et al., 1994), and then scraped and washed in cold buffer A and pelleted by cen- trifugation (Ross et al., 1997). The cells were resuspended in buffer A and ho- mogenized in a Dounce homogenizer. Homogenates were centrifuged as de- scribed above. The protein concentration of prepared extract was 5–10 g/ l. Neuronal cultures Primary cultures of embryonic chick forebrain neurons were generated as described previously (Zhang et al., 1999). Briefly, forebrains were dis- sected from 8-d chick embryos, trypsinized, dissociated, and plated on poly-L-lysine– (0.2 mg/ml, 16 h) and laminin- (0.02 mg/ml, 12 min) coated coverslips in MEM with 10% FBS for 2 h. Cells were inverted onto a mono- layer of chick astrocytes in N2-conditioned medium with serum (0.2% FBS) and cultured for 4 d at 37 C in 5% CO2. In vitro RNA transcription The pCZIP was constructed by inserting the 54 nucleotide zipcode of chick -actin mRNA into a pSP64-Poly(A) vector (Promega) at Hind III and Ava I sites. To construct the pCZIPm plasmid, the following pair of com- plementary oligos were synthesized, annealed, and inserted between Hind III and Ava I sites of pSP64 Poly(A) vector: Sense: 5 AGCTTACCGGACT- GTTACCATGTGTGTGTGTGCTGTGATGAAACAAAACCCATAAATGC 3 and antisense: 5 CCGGGCATTTATGGGTTTTGTTTCATCCAGCACACA- CACACATGGTAACAGTCCGGTA 3 . For gel mobility shift assays, [32P]-labeled RNA was generated by SP6 RNA polymerase directed in vitro transcription from Ava I linearized pCZIP DNA. The transcribed RNA was gel purified by 6%. For RNA affinity purification, polyadenylated transcripts were synthesized in vitro with SP6 polymerase (MEGAScript kit; Ambion) from EcoRI-linearized pCZIP con- structs. Trace amounts of [32P]-CTP were added to allow detection and quantitation of transcribed RNA. The transcribed RNA contained the 54 nts of pCZIP RNA and a poly(A) tail of 30 nts. Gel mobility shift assay and UV crosslinking Briefly, 105 CPM of the [32P]-labeled RNA probe was incubated at room temprature with 5 l of brain or fibroblast protein extract for 20 min in a 20- l binding solution containing 20 mM Hepes, pH 7.4, 50 mM KCl, 3 mM MgCl2, 2 mM DTT, and 5% glycerol. Unbound RNAs were degraded by a 10-min incubation with 1 U of RNase T1, and nonspecific RNA–pro- tein interactions were minimized by incubation with 5 mg/ml heparin for 10 min. The RNA–protein complexes formed were separated in a 4% na-