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...

Vgl mRNA, a maternal message encding a member of the transforming growth factor (3 superfamily, undergoes localization to the vegetal cortex of Xenopus laevis oocytes during a narrow period of oogenesis. A 340-nudweotde sequence has been identified in Vgl RNA that directs its vegetal localization [Mowry, K. L. & Melton, D. A. (1992) Science 255, [991][992][993][994]. To understand how cis-and trans-acting factors are involved in Vgl mRNA localization, we have looked for specific interactions in vitro between oocyte proteins and Vgl mRNA. S100 extracts of late-stage oocytes contain a proteinbinding activity that protects specific regions of labeled Vgl mRNA from degradation by RNase T1. The use of different regions of Vgl RNA in competition reactions reveals two binding sites, both in the first half of the 3' untranslated region of Vgl message. UV crosslinking predominantly labels a 69-kDa protein; saturation analysis and competitor studies indicate that this protein binds with a high affinity to the downstream site, which corresponds to the 340-nucleotide vegetal localization sequence. Binding to this region is inhibited by another vegetally localized message, transforming growth factor (35 but is not inhibited by an animally localized RNA, An2. These data indicate that vegetally localized mRNAs share a binding motif that helps them achieve their intracellular distribution through specific RNA-protein interactions.The polarity of Xenopus oocytes is evident from very early stages and is perhaps presaged by an asymmetry detectable in primordial germ cells (1, 2). As oogenesis proceeds, this polarity is elaborated by a series of morphological events that occur along the animal-vegetal axis, such as asymmetric distribution of yolk platelets, migration of pigment granules to the animal hemisphere, accumulation of polar granules at the vegetal pole, and the displacement ofthe germinal vesicle to the animal hemisphere (3,4). On a molecular level, this organization is reflected in a small class of RNA molecules that are localized to particular regions of the oocyte cytoplasm (5, 6). The heterogeneity of the single-cell egg is then translated into cellular differences in the developing embryo by early cleavage planes, helping to create the cell-cell interactions that contribute to producing the body axis. Despite the importance of polarity in oocytes, little is understood about its generation, molecular nature, or interpretation.Vgl mRNA, a member of the transforming growth factor ,8 (TGFB) superfamily, is localized to the vegetal cortex of late-stage oocytes and remains in the vegetal hemisphere of early embryos until after gastrulation (5, 7). Synthesized early during oogenesis and distributed homogeneously throughout stage I and II oocytes, Vgl message accumulates along the vegetal cortex of oocytes during a short period in the middle of oogenesis, where it remains until maturation (8-10). Localization of Vgl mRNA appears to occur in at least two steps, the first being translocation ofthe message to the vegeta...

Localized RNAs are found in a variety of somatic and developing cell types. In many cases, microtubules have been implicated as playing a role in facilitating transport of these RNAs. Here we report that Vg1 RNA, which is localized to the vegetal cortex of Xenopus laevis oocytes, is associated with microtubules in vivo. Because of the ubiquitous nature of tubulin, the association of specific RNAs with microtubules is likely to involve factors that recognize both RNA and microtubules. Vg1 RNA binding protein (Vg1 RBP), previously shown to bind with high affinity to the vegetal localization site in Vg1 RNA, appears to function in this capacity. Vg1 RBP is associated with microtubules: it is enriched in microtubule extracts of oocytes and is also co‐precipitated by heterologous, polymerized tubulin. Furthermore, Vg1 RBP binding activity is required for the specific association of Vg1 RNA to microtubules in vitro. These data suggest a general model for how specific RNAs can be localized to particular sites via common cytoskeletal elements.

The multistep pathway leading to intracellular RNA localization is known to involve cis-acting signals in targeted mRNAs, which are presumably recognized by specific RNA-binding proteins and interact with a functional cytoskeleton. Tau RNA is localized to the proximal hillock of rat axons, and this movement requires intact microtubules. Because Xenopus oocytes demonstrate a clear polarity involving microtubule-mediated RNA localization, we have studied the distribution of tau RNA injected into oocytes. We find that a fragment from the 3'-untranslated region of tau RNA is localized to the vegetal cortex of stage III/IV oocytes in a distribution indistinguishable from Vg1 RNA, a vegetally localized oocyte mRNA. A fragment from the tau RNA coding region, however, is homogeneously distributed in oocytes. Tau RNA contains a functional binding site for Vg1 RBP, a Xenopus microtubule-associated protein that binds vegetally localized oocyte RNAs with high affinity, and this binding correlates with vegetal localization ability. The present studies demonstrate, for the first time, localization of heterologous RNA in oocytes. Given the role of Vg1 RBP as a mediator of specific RNA-microtubule interactions, these results are strong evidence that Vg1 RBP is involved in the vegetal localization of RNAs in oocytes and raise the intriguing possibility of the existence of proteins with similar function in neurons.