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The 3' untranslated region of mRNA encoding PHAX, a phosphoprotein required for nuclear export of U-type snRNAs, contains cis-acting sequence motifs E2 and VM1 that are required for localization of RNAs to the vegetal hemisphere of Xenopus oocytes. However, we have found that PHAX mRNA is transported to the opposite, animal, hemisphere. A set of proteins that cross-link to the localization elements of vegetally localized RNAs are also cross-linked to PHAX and An1 mRNAs, demonstrating that the composition of RNP complexes that form on these localization elements is highly conserved irrespective of the final destination of the RNA. The ability of RNAs to bind this core group of proteins is correlated with localization activity. Staufen1, which binds to Vg1 and VegT mRNAs, is not associated with RNAs localized to the animal hemisphere and may determine, at least in part, the direction of RNA movement in Xenopus oocytes.
FIGURE 1. PHAX mRNA is localized to the animal hemisphere of Xenopus oocytes. (A) A portion of the Xenopus EST AW766532 is shown, highlighting the binding site for VgRBP71 (red) and a downstream polyadenylation signal (green). The sequence at the binding site for VgRBP71 (bold, underlined) in the EST is compared with that in Vg1 mRNA. (B) Oocytes were separated according to developmental stage (I–VI) and total RNA isolated for Northern blot analysis. Each lane contains 10 oocyte equivalents of RNA. Complementary probes for PHAX and GAPDH (loading control) mRNAs were used simultaneously. The positions of size standards (nucleotides, nt) are indicated. (C) Scheme showing the relative positions of E2 and VM1 motifs, VgRBP71 binding sites, and polyadenylation signals in the 3′ UTRs of Vg1, VegT, and PHAX mRNAs. (D) Immunohistochemical staining of stage V/VI oocytes following in situ hybridization with digoxigenin-labeled probes for Vg1 mRNA (left panel) and PHAX mRNA (right panel). The discernible indentation on the oocyte surface acts as a marker for the animal hemisphere.
FIGURE 2. RNAs localized to either the vegetal or animal hemispheres are cross-linked to the same set of proteins. Internally radiolabeled PHAX (left), Vg1 (center), or An1 (right) RNA was added to whole-cell extract prepared from mixed-stage oocytes. Following UV irradiation, samples were treated with ribonuclease and then analyzed by SDS electrophoresis followed by autoradiography. A 20-fold excess of unlabeled, competitor RNA was added to individual samples, as indicated below each lane. Nonspecific RNA (ns) is a 356-nt transcript from the vector pBluescript KS.
FIGURE 3. Functional localization signals extend throughout the length of An1 mRNA. (A) Scheme showing the location of E2 and VM1 motifs in the An1 3′ UTR targeted for mutagenesis (left). Empty symbols represent sites of mutation. Localization phenotypes of injected RNAs (right). Following immunohistochemical staining of oocytes injected with DIG-labeled RNA, each was visually judged for localization. Representative oocytes injected with each RNA are shown along the side of the table oriented with the animal hemisphere upward. (B) UV cross-linking and localization assays of Vg1 and An1 mRNAs. Full-length Vg1 mRNA (lane 1) is cross-linked to the six core group of proteins. Deletion of the Vg1 3′ UTR, which removes the VLE, eliminates cross-linking of the binding proteins (lane 2) and abolishes localization of the RNA. Deletion of the An1 3′ UTR (lane 3) or the 3′ UTR and the last 850 nt of the ORF (lane 4) do not eliminate cross-linking activity or localization of these mutant RNAs.
FIGURE 4. Staufen1 is associated only with RNA localized to the vegetal hemisphere. Staufen1 was immunoprecipitated from whole-cell extract prepared from stage III/IV oocytes. The associated RNA was reverse-transcribed into cDNA and amplified by PCR using gene-specific primers (indicated above each panel). (Lanes 1) PCR standards generated using total oocyte RNA as a template; (lanes 2) control precipitation with protein A-Sepharose beads in the absence of antibody; (lanes 3) precipitation from extract incubated with Staufen1 antibody. Ribosomal protein L5 and actin are not localized mRNAs and serve as negative controls.
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