The highly conserved VICKZ (Vg1 RBP/Vera, IMP-1,2,3, CRD-BP, KOC, ZBP-1) family of RNA-binding proteins recognize specific cis-acting elements in a variety of different RNAs and have been implicated in cell polarity and migration, cell proliferation, and cancer. In just the last two years, the use of transgenic mice, antisense RNA, and RNAi (RNA interference) techniques have contributed to our understanding of the roles of these proteins and how they interface with many diverse processes in cells. In this article, I will review the recent advances relating to VICKZ proteins and try to suggest a framework for understanding how, in conjunction with other RNA-binding proteins, they participate in a combinatorial fashion to help determine the fate of their RNA targets and, ultimately, the function of cells in which they are expressed. Such a 'post-transcriptional operon' model, as proposed by Keene and Tenenbaum [(2002) Mol. Cell 9, 1161-1167], can explain the differential, integrated action of multiple RNA-binding proteins on mRNA targets.

Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3 UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of ␤ actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting. Received January 21, 1998; revised version accepted March 25, 1998. Intracellular RNA localization leads to asymmetric protein synthesis, a necessary step in the process of pattern formation during early embryogenesis in many species (for review, see St. Johnston 1995;Gavis 1997). In Xenopus oocytes, morphological and molecular differences between the animal and vegetal hemispheres help define the primary axis around which subsequent development proceeds. Two temporally distinct pathways have been identified for vegetal RNA localization. The early pathway facilitates localization of several RNAs found to be localized during stages I-II of oogenesis (Xcat-2, Xlsirts, Xwnt-11), appears to be microtubule-independent, and is correlated with the migration of the mitochondrial cloud to a small region at the vegetal pole (Kloc et al. 1993;Mosquera et al. 1993;Forristall et al. 1995;Kloc and Etkin 1995;Zhou and King 1996a). The second pathway occurs during late stage III-early stage IV, requires intact microtubules, and targets RNA to a tight shell along the entire vegetal cortex (Melton 1987;Yisraeli and Melton 1988;Yisraeli et al. 1990). So far, only one mRNA, Vg1, is known to localize via the second pathway in vivo, although Xcat-2 RNA can employ this pathway when injected into stage III oocytes (Zhou and King 1996b). VegT/Xombi/Apod/Brat RNA is also localized to the vegetal cortex in a manner resembling the second pathway, but it is not yet known whether this localization requires intact microtubules; furthermore, it is released from the cortex in stage V/VI oocytes, significantly earlier than is Vg1 RNA (Lustig et al. 1996;Stennard et al. 1996;Zhang and King 1996). Cis-acting elements mediating one or the other pathway have been mapped, by deletion analysis, to extensive regions of the 3Ј UTRs (Mowry and Melton 1992; Zhou and King 1996a,b; Gautreau et al. 1997). Trans-acting factors that bind these regions and may provide a link between the RNA and components of the cytoskeleton have been characterized only preliminarily, and the connection between these interactons and localization is just beginning to be defined (Schwartz et al. 1992;Mowry 1996;Deshler et al. 1997).Vg1 RNA-binding protein (RBP) is a 69-kD oocyte protein that binds specifically t...

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The effect of DNA methylation on the transcriptional activity of the hamster adenine phosphoribosyltransferase (aprt) and the herpes thymidine kinase (tk) genes has been investigated. By using M13 constructs containing these gene sequences, specific segments of each gene were methylated in vitro by restriction fragment primer-directed second-strand synthesis using the substrate 2'-deoxy-5-methylcytidine triphosphate (dmCTP). These hybrid-methylated molecules were inserted into mouse Ltk-cells by DNA-mediated cotransfer. In all cases, the integrated sequences retained the in vitro-directed methylation pattern. The aprt gene was inhibited by CpG methylation in the 5' region but was unaffected by methylation at the 3' end or in adjacent M13 sequences. In contrast to this, DNA methylation in both the 5' promoter region and the 3' structural region of the tk gene had a strong inhibitory effect. This suggests that this modification may affect transcription by mechanisms that do not involve the direct alteration of recognition sequences for RNA polymerase. MATERIALS AND METHODSM13 Constructs and Synthesis. The M13 clones containing the 2.0-kilobase (kb) Pvu II herpes tk fragment (9) were obtained from S. McKnight and those containing the 3.8-kb BamHI hamster aprt fragment (10) were made by D. Littman and kindly provided by him for these experiments. Singlestranded DNA was prepared (11) from these recombinant phages by polyethylene glycol precipitation followed by standard DNA extraction techniques. The plasmid pTM (12), containing the bacterial agpt gene with the tk promoter region, was obtained from R. Flavell and was prepared by the method of Clewell (13).Second-strand synthesis of single-stranded M13 molecules was performed under conditions described (14) for 1 hr at 15°C using a DNA concentration of 2-3 ,g per 100 ,ul and a polymerase concentration of 75 units/ml. The 15-mer universal M13 primer was obtained from New England Biolabs and was used at a level of 25 ng per pg of M13 DNA. Under these conditions, the synthesis reaction went to 90%-95% completion, as judged by gel electrophoresis analysis, but it did not produce strand displacement. These reactions were carried out using either dCTP or 2'-deoxy-5-methylcytidine triphosphate (dmCTP) (P-L Biochemicals) to obtain methylated molecules and their unmethylated controls. Restriction fragment primers were isolated from low-melting agarose gels and were hybridized to M13 DNA in 0.1 M NaCl at 60°C after denaturation for 3 min at 100°C in 1 mM EDTA. The primer was used at a 5-fold molar excess to get optimum use of the template. Double-stranded molecules were gel-purified before their introduction into L cells by DNA-mediated gene transfer. The synthesis reaction was sometimes performed using a small amount of radioactive nucleotides to test these molecules for strand displacement by specific restriction enzyme analysis (Fig. 1A).Analysis of Transfected Cells. DNA-mediated gene transfer into mouse Ltk-cells was performed as described (15) (22) and were hybri...

Mouse teratocarcinoma cells induced to differentiate in vitro undergo a massive (30%) demethylation of DNA. A similar undermethylation is also observed in the mouse extraembryonic membranes, the yolk sac and placenta. In both cases, the decrease in methyl moieties occurs at a large number of CpG sites spread out over the entire genome, as indicated by a restriction enzyme analysis of several mouse genes including dhfr, 13-major globin, and the H-2K gene family. In contrast to this, the embryo itself appears to undergo methylation de novo during early stages of embryogenesis. Thus, as opposed to somatic cells, events during early mouse development are associated with wide variations in the level of DNA methylation. Although these changes in DNA methylation seem to be an integral part of the differentiation process, its relation to specific gene expression is still unclear.

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After mid-blastula transition, populations of cells within the Xenopus embryo become motile. Using antisense morpholino oligonucleotides, we find that Vg1 RBP, an RNA-binding protein implicated in RNA localization in oocytes, is required for the migration of cells forming the roof plate of the neural tube and, subsequently, for neural crest migration. These cells are properly determined but remain at their site of origin. Consistent with a possible role in cell movement, Vg1 RBP asymmetrically localizes to extended processes in migrating neural crest cells. Given that Vg1 RBP is a member of the conserved VICKZ family of proteins, expressed in embryonic and neoplastic cells, these data shed light on the likely role of these RNA-binding proteins in regulating cell movements during both development and metastasis.

The animal and vegetal ends of Xenopus oocytes have distinctly different developmental fates. At the molecular level, several maternal mRNAs have been isolated that are localized to either the animal or vegetal hemisphere. One of these mRNAs, Vg1, is distributed homogeneously throughout the cytoplasm of early-stage oocytes and gets localized during oogenesis to a tight shell at the vegetal cortex of middle and late-stage oocytes. We have used an in vitro culture system to demonstrate that exogeneous Vg1 mRNA injected into middle-stage, but not late-stage, oocytes gets localized in a similar fashion to the endogenous message. Furthermore, translation of Vg1 mRNA is not required for the localization of the message itself. These results show that the information necessary to interpret the animal-vegetal polarity in oocytes is present in the naked mRNA transcript.