RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization

Abstract

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...

H19 RNA Binds Four Molecules of Insulin-like Growth Factor II mRNA-binding Protein

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Vasopressin mRNA localization in nerve cells: Characterization of cis-acting elements and trans-acting factors

“…However, the data do not rule out the possibility that the actin-based cytoskeleton may be necessary for mRNA anchoring within the neurites. Recently, evolutionarily conserved RNAbinding proteins have been identified that may play a role in transporting ␤-actin transcripts in fibroblasts (27) and vegetal pole 1 (Vg1) mRNA in Xenopus oocytes (28,29), respectively. Although ␤-actin mRNA is transported along microfilaments, microtubules are prerequisite for Vg1 mRNA sorting to the vegetal hemisphere.…”

Cloning and characterization of a novel 90 kDa ‘companion’ auto-antigen of p62 overexpressed in cancer

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