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In Xenopus laevis, maternal transcripts that localize to the vegetal cortex of the oocyte are specifically inherited by prospective germ cells during cleavage stages. While a large fraction of maternal transcripts is degraded during the maternal to zygotic transition (MZT), transcripts associated with the germ-line are stable. A sequence in the dead end 1 3'UTR mediates vegetal localization in the oocyte as well as miR mediated clearance in somatic cells and germ cell specific stabilization during the MZT in embryos. We could identify Tia1 to co-precipitate with known components of vegetal localization RNPs in X. laevis oocytes. Tia1 interacts and co-localizes with various localization elements from vegetally localizing RNAs. In X. laevis embryos, ectopic expression of Tia1 counteracts somatic degradation of dnd1 localization element reporter RNAs and it can synergize with Dnd1 protein in reporter RNA stabilization. Ectopic Tia1 also protects several endogenous localizing and germ cell specific mRNAs from somatic degradation. Thus, proteins that protect germ-line transcripts from miR mediated decay during the MZT in embryos might bind these RNAs already in the oocyte.
Fig 1. Tia1 co-precipitates known localization RNP complex components in Xenopus oocytes. Flag-tagged versions of different localization proteins and Tia1 were expressed in Xenopus oocytes by means of RNA micro-injection and analyzed by Western blot for co-precipitation of known localization factors in the absence or presence of RNase A. Celf1 and Ptbp1 served as controls for known localization complex components. Immunoprecipitation with extract from uninjected oocytes served as a negative control. The asterisk indicates an unspecific signal caused by cross-reaction of the anti-Flag antibody.
Fig 2. Tia1 is predominantly cytoplasmic and co-localizes with dnd1-LE-RNA at the vegetal cortex in Xenopus oocytes. (A) Temporal analysis of Tia1 expression during Xenopus oogenesis and embryogenesis. Western blot analysis of Tia1 with equivalent amounts of oocyte and embryonic extracts; stages of oogenesis and embryogenesis were as indicated. (B) Western blot analysis of Tia1 with cytoplasmic (C) and nuclear (N) fractions from staged oocytes. Igf2bp3 served as control for a cytoplasmic protein and Hnrnpab was utilized as control for a predominantly nuclear protein. (C) Co-localization analysis of endogenous Tia1 protein and vegetally localizing, microinjected Cy3-dnd1-LE in Xenopus oocytes. Tia immunostaining was performed on Cy3-dnd1-LE RNA injected stage III oocytes. Scale bars indicate 100 mm (whole oocyte) and 20 mm (magnification).
Fig 3. Tia1 binds to the 5’ region of the dnd1-LE. (A) In vitro interaction of Flag-tagged Tia1 with different LE RNAs. Cy3-labeled LE RNAs and b-globin-3’UTR control RNA were co-immunoprecipitated with Flag-tagged, in vitro translated Tia1, Celf1 or Ptbp1. Non-programmed reticulocyte lysate served as a negative control (-). Supernatant and bound RNAs were separated by UREA-PAGE and detected by fluorescence imaging. (B) In vitro interactions of Flag-tagged Tia1 with Cy3-labeled full length (FL) and 5´- or 3´-deleted fragments of the dnd1-LE RNA. The region critical for Tia1 binding is marked in light grey (nt 40-106).
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4. Mutation of the uracil-rich 5’region in the dnd1-LE reduces Tia1 binding. (A) Sequence of full length wild-type (wt) and mutant (mut) dnd1-LE. Multiple uracil bases in the Tia1 binding site (boxed) were replaced by adenines by site-directed mutagenesis (red letters). The miR-18 target is shown in blue. (B) Binding of bacterially expressed Tia1 to wild-type (wt) and mutant (mut) dnd1-LE was analyzed by electrophoretic mobility shift assays (see Figure S2B). The relative amount of complexed RNA is plotted against the individual protein concentrations using non-linear curve fitting; approximate KD values for wild-type and mutant dnd1-LE RNAs are indicated.
Fig 5. Somatic expression of Tia1 stabilizes injected dnd1-LE reporter RNA and endogenous localizing mRNAs. (A) Wild-type (wt) or mutant (mut) dnd1-LEreporter (dnd1-LE-R) RNAs containing the gfp open reading frame were injected into both vegetal blastomeres of 2-cell stage Xenopus embryos. At stage 32, whole mount in situ hybridization was performed against gfp. (B) Embryos injected with wildtype or mutant dnd1-LE-reporter RNAs alone or co-injected with 400 pg RNA encoding Tia1. C) Quantification of dnd1-LE-reporter RNA levels as scored in the injected embryos. Mean values of 2 independent experiments are shown. Error bars indicate the standard error of the mean. (D) Fold change in RNA levels of different endogenous localizing and control mRNAs at the developmental stages indicated after overexpression of Tia1. Xenopus 2-cell stage embryos were injected with 200 or 400 pg tia1 RNA. Embryos were grown until stages 8, 11 or 14 and subjected to total RNA extraction. RNA samples were analyzed using the nCounter® Gene Expression assay (NanoString Technologies). The averaged fold change of selected RNAs over uninjected control embryos of two independent experiments are shown, error bars indicate the standard error of the mean.
Fig 6. Tia1 synergizes with Dnd1 in the stabilization of dnd1-LE-reporter RNA in Xenopus embryos. (A) Embryos injected with gfp-dnd1-LE reporter RNA (dnd1-LE-R wt) alone or co-injected with low doses of RNA encoding either Tia1 or Dnd1 or both. Reporter RNA levels were detected by WMISH at stage 32. Injected wild-type dnd1-LE reporter RNA alone is degraded in the soma and stable in PGCs. Over-expression of low doses of Tia1 or Dnd1 does not stabilize dnd1-LE reporter RNA in the soma. Co-expression of same doses of Tia1 and Dnd1 leads to somatic stabilization of dnd1-LE reporter RNA. (B) Quantification of dnd1-LE reporter RNA levels scored in the injected embryos. Mean values of three independent experiments are shown, error bars indicate the standard error of the mean.
Fig S1. In vitro translated Flag-tagged proteins used for co-immunoprecipitations. In vitro translated Flag-tagged Tia1, Celf1 and Ptbp1 were separated by SDS-PAGE and detected by a-Flag Western blotting. Non-programmed reticulocyte lysate served as negative control (-). (A) Protein control for RNA Co-IP of different RNA-LEs (Figure 3A). (B) Protein control for RNA-Co-IP of 5’deleted dnd1-LE fragments (Figure 3B). (C) Protein control for RNA-Co-IP of 3’deleted dnd1-LE fragments (Fig 3B).
Fig S2. Binding of recombinant Tia1 to dnd1-LE requires uracil rich 5’region. (A) Recombinant Tia1 used for EMSAs. The protein quantity was estimated by Bradford assay and comparison to BSA signals of known quantity. (B) Representative mobility shift analyses of bacterially expressed Tia1 and Cy3-labeled RNAs, used for quantification of Tia1 binding to wild-type (wt) and mutant (mut) dnd1-LE.
