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Fig. 1. Expression pattern of venus fused with region A of DEADSouth 3′ UTR in the middle of 3′ UTR of Xenopus β-globin (A and A′) and the sequence of region A (B). (A) Whole-mount lateral view of stage 41 tadpole. Scale bar: 1 mm. (A′) High-magnification of the area indicated in (A). Arrowheads indicate PGCs. Scale bar: 250 μm. (B) The possible target sites for miR-427 (MRE, in red) found in the region A of DEADSouth 3′ UTR (DDBJ/GenBank/EMBL accession No. AF190623 nucleotides 1804–2287).
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Fig. 2. DEADSouth mRNA was degraded specifically by miR-427. (A) RT-PCR for v-DS mRNA in embryos at stages 11 and 30 coinjected with anti-miR427 LNA or anti-miR427mut LNA. The ubiquitously expressed ODC gene was used as a control. Note that the injected v-DS mRNA was detected in every embryo at stage 11, but detected only in embryos coinjected with anti-miR427 LNA at stage 30. (B) Expression of venus reporter mRNA at stage 30. (C) and (D) Expression of venus reporter mRNA at stage 30 in an embryo coinjected with anti-miR427 LNA or anti-miR427mut LNA. Note that Venus fluorescence was detected only in the embryos coinjected with anti-miR427 LNA. Scale bar, 1 mm.
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Fig. 3. Degradation of DEADSouth mRNA via MREs in region A of the 3′ UTR. (A) Sequences of wild type (upper) and mutated MREs (lower). Three possible MREs in region A were mutated (MRE1m2m3m). (B–G) MRE123 or MRE1m2m3m reporter mRNA was injected without/with siR-427 or siR-427m into the animal hemisphere of fertilized eggs. The embryos at 41 were examined for degradation of injected mRNA by Venus fluorescence. Note that the MRE1m2m3 m reporter mRNA was degraded specifically by siR-427m, but not by siR-427. Scale bar, 1 mm.
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Fig. 4. Degradation of reporter mRNA with normal or mutated MREs. (left) Northern blot analysis for total RNA (10 μg/lane) at stages 1 (just after microinjection), 11 (gastrula) and 30 (tailbud). 18S rRNA bands are shown as loading controls (lower in each row). Closed and open arrowheads indicate sizes of the original and elongated mRNA, respectively. The ratios of band intensity at stage 30 to that at stage 11 are also shown. Each ratio was the mean of duplicated experiments. (right) WISH for venus mRNA in stage 30 embryos injected with the indicated mRNA. The percentage of embryos in which a Venus fluorescence signal was detected in the somatic cells is shown in the right. The lowest two rows show the results from the experiments in which MRE123 and MRE1m2m3m mRNAs were injected at the vegetal pole, respectively. Scale bar, 1 mm.
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Fig. 5. Zygotic expression of DEADSouth gene. (A) Sequence alignment of the 3′ UTR of DEADSouth cDNA of X. laevis (DDBJ/GenBank/EMBL accession No. AF190623 nucleotides 1625–1892) and X. borealis. PCR primers are indicated with arrows. PCR using these primers resulted in species-specific amplification of 248 and 224 bp sequences from X. laevis and X. borealis, respectively. (B) Species-specific amplification using cDNA from X. laevis or X. borealis oocytes as a template. (C) Expression of DEADSouth gene in hybrid embryos between X. laevis and X. borealis at stages 3 and 20. X. laevis- and X. borealis-specific amplifications are indicated by open and closed arrowheads, respectively, in (B) and (C). Note that the DEADSouth transcript from the male was detected only in stage 20 embryos.
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Fig. 6. Expression of miR-427 during early development in X. laevis. WISH with antisense (A–H) and sense LNA probes (I) for miR-427. Signals in blue (high) to brown (low) depended on the expression level. (A–C) Animal, lateral and vegetal views at stage 9, respectively. (D, E) High magnification images of the equatorial region of the stage 9 embryo. Nuclei are also stained with PI in (E). (F) Vegetal views at stage 11. (G) Dorsal view at stage 17. (H) (I) Lateral views at stages 31 and 9, respectively. (J, K, L) Vegetal views of WISH at indicated stages with antisense Xpat riboprobe (red) and antisense miR-427 LNA probe (blue). Note that signals for Xpat are not overlapped with those for miR-427. Scale bars are 0.5 mm in (A–C, F, G, I), 0.1 mm in (D, E), 0.2 mm in (J–L) and 1 mm in (H), respectively. (M) Northern blot analysis for miR-427. Total RNAs from whole embryos (0.5 μg), endodermal cells (1268 cells) and PGCs (1268 cells) at stage 17 were loaded into lanes 1, 2 and 3, respectively. After detection for miR-427 (upper), the blot was reprobed with a 5S rRNA probe for normalization (lower).
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Fig. 7. Effect of siR-427 on the degradation of reporter mRNA in PGCs of stage 41 tadpoles. (A–D) Whole-mount lateral views are shown with injected reporter mRNA and siRNA. (A′–D′) High-magnification of the areas including aligned PGCs indicated in (A–D), respectively. Scale bars, 1 mm in (D) and 500 μm in (D′).
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Supplementary Figure 1. Degradation of DEADSouth mRNA was associated with deadenylation. (A) Poly(A) profiles of injected reporter mRNAs βG/ABC and MRE123m. Total RNA from the injected embryos at the indicated stages was subjected to PAT assay.
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Supplementary Figure 2. Effect of siR-427 on the degradation of reporter mRNA in PGCs. (AâL) Dissociated endodermal cell masses are shown from stage 20-embryos injected with MRE123 plus Alexa594-labeled siR-427m (AâC), MRE123 only (DâF), MRE1m2m3m plus Alexa594-labeled siR-427m (GâI) and MRE1m2m3m only (JâL). The signals for siR-427m (purple) and Venus (green) overlap partially as indicated by white arrowheads. Scale bar, 250 μm.
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