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In zebrafish oocytes, cyclin B1 mRNAs are transported to the animal polar cytoplasm. To elucidate the molecular basis of cyclin B1 mRNA transport, we analyzed zebrafish Staufen1, a protein known to play a central role in mRNA transport to the vegetal pole of Xenopus oocytes. Zebrafish Staufen1 interacts with cyclin B1 mRNA throughout oocyte growth. Both cyclin B1 mRNA and Staufen1 are evenly distributed in the cytoplasm of young oocytes but are co-localized to the animal polar cytoplasm in later stages. Real-time imaging showed that the plus ends of oocyte microtubules are free in the cytoplasm in early stages but anchored to the animal polar cytoplasm in later stages. Transport of cyclin B1 reporter mRNA to the animal polar cytoplasm was inhibited by disruption of microtubules and injection of antibodies against Staufen1 or Kinesin1, a plus-end-directed microtubule motor that interacts with Staufen1, indicating that the transport depends on movement along microtubules toward the plus ends. Reporter mRNAs with an element required for the vegetal localization of vg1 mRNA in Xenopus oocytes were localized to the animal polar cytoplasm in zebrafish oocytes, indicating that the element is functional for animal polar localization in zebrafish oocytes. Our findings suggest that cyclin B1 mRNA-Staufen1 protein complexes are transported toward the animal pole of zebrafish oocytes by the plus-end-directed motor protein Kinesin1 along microtubules and that a common mRNA transport machinery functions in zebrafish and Xenopus oocytes, although its transport direction is opposite due to different organizations of microtubules.
Fig. 1. Staufen1 protein (Stau1) during zebrafish oocyte growth. (A) Oocyte extracts from stages I to IV were analyzed by immunoblotting with anti-Stau1 (GP) and anti-α-Tubulin antibodies. (B) Stage IV oocytes overexpressed with (+) or without (−) Flag-Stau1 were immunoprecipitated with (+) or without (−) anti-Flag M2 antibody (Flag IP). Flag-Stau1 protein and cyclin B1 mRNA in the immunoprecipitates were detected by immunoblotting with anti-Flag M2 antibody and RT-PCR, respectively. (C) Extracts from stage IV oocytes were treated with (+) or without (−) RNase A and immunoprecipitated (IP) with anti-Pum1 (Pum1), anti-Stau1 (Stau1) or control (−) antibodies. Pum1 and Stau1 in the immunoprecipitates were detected by immunoblotting. (D) Experiments similar to those in (C) using anti-IMP3 and anti-Stau1 antibodies. (E) Extracts from stage I to IV oocytes were immunoprecipitated (Stau1 IP) with anti-Stau1 (+) or control antibodies (−) and cyclin B1 mRNA in the immunoprecipitates was detected by RT-PCR. Extracts from stage IV oocytes before immunoprecipitation were also subjected to RT-PCR to confirm the presence of cyclin B1 mRNA in the original extracts (initial).
Fig. 2. Distribution of cyclin B1 mRNA and Stau1 protein during zebrafish oocyte growth. Paraffin sections of zebrafish oocytes were simultaneously analyzed by in situ hybridization and immunostaining. Hoechst 33258 staining of DNA (blue: C, D), in situ hybridization of cyclin B1 mRNA (green: A1; B1; C1, 4; D1, 4), immunostaining of Stau1 (red: A2; B2; C2, 5; D2, 5). and their merged and magnified images (A3; B3; C3, 6; D3, 6) are shown. CA, cortical alveolus; EC, egg chorion; FC, follicle cell; GV, germinal vesicle; MP, micropyle; YG, yolk granule. Scale bars, 100 μm. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
Fig. 3. Effects of 3′ UTR sequences on the gfp-cyclin B1 reporter mRNA localization to the animal polar cytoplasm. (A) Structures of reporter mRNAs. The cyclin B1 3′ UTR sequence was replaced with either Xenopus vg1/vegetal localization element (VLE) or SV40 polyadenylation signal (SV40) sequences. (B) Reporter mRNAs were detected by whole-mount in situ hybridization using a gfp probe, and the resulting signals were classified into 5 groups (Aggregated, Class I, II, III and Not localized: Class I, signal restricted to less than 1/4 of the oocyte hemisphere; class II, signal ranging from 1/4 to 1/2 of the hemisphere; and class III, signal expanding over 1/2 of the hemisphere [6]). Localization of endogenous cyclin B1 mRNAs is also shown (Wild type). Arrows indicate localized signals. Scale bars, 100 μm. (C) The numbers of oocytes showing each localized signal were counted. Data were collected from three independent experiments. The numbers in parentheses indicate the total numbers of oocytes analyzed. (D) Extracts from oocytes injected with reporter constructs (3′ UTR, VLE, SV40) were subjected to immunoprecipitation with (+) or without (−) anti-Stau1 antibody. The reporter mRNAs in the immunoprecipitates (Stau1 IP) and extracts before immunoprecipitation (initial) were detected by RT-PCR using the gfp sequence.
Fig. 4. Distribution of EB1 in zebrafish stage IV oocytes revealed by in situ hybridization of cyclin B1 mRNA (green), immunostaining of EB1 (red) and merged images. The animal (upper box) and vegetal (lower box) polar regions in (A) are magnified in (B) and (C), respectively. Hoechst 33258 staining of DNA is shown in blue. Broken lines indicate the boundary between the oocyte and follicle layer (B, C). Scale bars, 100 μm (A), 50 μm (B, C). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
