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Siah proteins are vertebrate homologs of the Drosophila 'seven in absentia' gene. In this study, we characterized two splicing forms, Siah1a and Siah1b, of the Xenopus seven in absentia homolog 1 gene (Siah1). Overexpression of xSiah1a led to severe suppression of embryo cleavage, while that of xSiah1b was not effective even at a high dose. Competition analysis demonstrated that co-expression of xSiah1a and 1b generated the same phenotype as overexpression of xSiah1a alone, suggesting that xSiah1b does not interfere with the function of xSiah1a. Since xSiah1b has an additional 31 amino acids in the N-terminus compared to xSiah1a, progressive truncation of xSiah1b from the N-terminus showed that inability of xSiah1b to affect embryo cleavage was associated with the length of the N-terminal extension of extra amino acids. The possible implication of this finding is discussed.
Fig. 1.
Sequence comparison of the alternative splicing variants of Siah1 among human and frog. hSiah1L (EAW82726), hSiah1b (NP_001006611), hSiah1a (NP_003022), xSiah1a and xSiah1b.
Fig. 2.
Temporal expression of xSiah1a and xSiah1b during Xenopus laevis embryogenesis. (A) Schematic diagram of xSiah1a and xSiah1b in the genome. Positions of the primers in the Siah1 gene used for RT-PCR are indicated by arrows. UTR, untranslated region; CR, coding region. (B) RT-PCR analysis of xSiah1a and xSiah1b during Xenopus laevis embryogenesis. UE, unfertilized eggs, RT−, negative control, ODC, ornithine decarboxylase as a loading control.
Fig. 3.
xSiah1a suppresses Xenopus embryocleavage through a proteosome dependent degradation pathway. (A) Overexpression of xSiah1a suppresses Xenopus embryocleavage in a dose-dependent manner. (B) Two hundred picograms xSiah1a mRNA injection compared to 2 ng xSiah1b mRNA injection. (C) Schematic representation of the construct of xSiah1a-2A-GFP. (D) Expression of XSiah1a was suppressed by MG132, a proteosome inhibitor. MG132 was injected to embryos after injection of 500 pg of xSiah1a-2A-GFP mRNA. (E) Overexpression of xSiah1a formed multinuclear cells. Red arrow shows an enlarged nucleus; yellow arrow indicates a normal nucleus. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4.
Analysis of xSiah1b function. (A) Co-injection of xSiah1a and xSiah1b. Two nanograms of xSiah1b mRNA was co-injected with 100 and 200 pg xSiah1a mRNA, respectively. (B) Diagram of the constructs of xSiah1b with different additional N-terminal truncations. (C) Injection of 50 pg of mRNA. (D) Western blot analysis of xKid expression to detect xSiah1a and xSiah1a activity in 293T cells. Equivalent amounts of xSiah1a, xSiah1b and pCS2 (250 ng), were co-transfected with Flag-xKid (1 μg).
