JCB Article A predominantly nuclear protein affecting cytoplasmic localization of -actin mRNA in ﬁbroblasts and neurons Wei Gu,1 Feng Pan,1 Honglai Zhang,2 Gary J. Bassell,2 and Robert H. Singer1 1Departments of Anatomy and Structural Biology and Cell Biology and 2Department of Neuroscience, Albert Einstein College of Medicine, Bronx, NY 10461 Tof chicken ﬁbroblasts and neurite growth cones of he localization of -actin mRNA to the leading lamellae developing neurons requires a 54-nt localization signal (the zipcode) within the 3 untranslated region. In this study we have identiﬁed and isolated ﬁve proteins binding to the zipcode. One of these we previously identiﬁed as zipcode binding protein (ZBP)1, a 4-KH domain protein. A second is now investigated in detail: a 92-kD protein, ZBP2, that is especially abundant in extracts from embryonic brain. We show that ZBP2 is a homologue of the human hnRNP protein, KSRP, that appears to mediate pre- mRNA splicing. However, ZBP2 has a 47–amino acid (aa) sequence not present in KSRP. Various portions of ZBP2 fused to GFP indicate that the protein most likely shuttles between the nucleus and the cytoplasm, and that the 47-aa insert promotes the nuclear localization. Expression of a truncated ZBP2 inhibits the localization of -actin mRNA in both ﬁbroblast and neurons. These data suggest that ZBP2, although predominantly a nuclear protein, has a role in the cytoplasmic localization of -actin mRNA. Introduction In diverse cell types, asymmetric localization of specific mRNAs generates cell polarity by controlling the translation sites and restricting the target proteins to the appropriate subcellular compartments (Bassell et al., 1999; Bassell and Singer, 1997, 2001; Jansen, 2001). During oogenesis in Drosophila and Xenopus, polarized localization of maternal mRNAs and consequent protein synthesis establish embry- onic patterning (Bashirullah et al., 1998; Deshler et al., 1998; Ephrussi et al., 1991). In yeast Saccharomyces cerevi- siae, mating-type switching is regulated by targeting ASH1 mRNA to the bud tip, where localized expression of Ash1p represses the HO endonuclease in the daughter cell (Amon 1996; Long et al., 1997; Takizawa et al., 1997). Asymmetrical segregation of mRNAs in subcellular locations is also observed in somatic cells, for example, MBP mRNA in oligodendrocytes (Ainger et al., 1993, 1997), MAP2 and tau mRNA in neurons (Kleiman et al., 1990; Litman et al., 1993; Behar et Address correspondence to Robert H. Singer, Dept. of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: (718) 430-8646. Fax: (718) 430- 8697. E-mail: rhsinger@aecom.yu.edu W. Gu and F. Pan contributed equally to this work. Key words: RNA localization; RNA binding proteins; nuclear-cytoplas- mic trafﬁcking; RNA splicing; KH domain proteins  The Rockefeller University Press, 0021-9525/2002/1/41/11 $5.00 The Journal of Cell Biology, Volume 156, Number 1, January 7, 2002 41–51 http://www.jcb.org/cgi/doi/10.1083/jcb.200105133 al., 1995), -actin mRNA in fibroblasts (Kislauskis et al., 1993), and neurons (Bassell et al., 1998; Zhang et al., 1999). The localization of -actin mRNA in motile fibroblasts and growth cones of developing neurons provides good models by which to understand the molecular mechanism whereby specific mRNAs are transported and targeted to precise cytoplasmic environments, hence promoting cellular asymmetry. Spatially restricted synthesis of actin proteins results from targeting of -actin mRNA at the leading edge of chicken embryonic fibroblasts (CEFs)* where actin poly- merization drives cell motility (Kislauskis et al., 1994). Fibroblasts with localized -actin mRNA migrate signifi- cantly further than those with nonlocalized -actin mRNA (Kislauskis et al., 1997). In cultured rat and chicken devel- oping neurons, the sorting of -actin mRNA to neurite growth cones has also been observed (Bassell et al., 1998; Zhang, et al., 1999), and mRNA localization is necessary for enrichment of -actin protein and forward movement of growth cones (Zhang et al., 2001). These data suggest that *Abbreviations used in this paper: aa, amino acid(s); CEF, chicken em- bryonic fibroblast; EGFP, enhanced green fluorescence protein; NLS, nuclear localization signal; RACE, rapid amplification of cDNA ends; UTR, untranslated region; ZBP, zipcode binding protein. 41