P-element Somatic Inhibitor Protein Binding a Target Sequence in dsx Pre-mRNA Conserved in Bombyx mori and Spodoptera litura
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Abstract:
Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.Keywords:
Doublesex
Spodoptera litura
Bombyx mori doublesex (Bmdsx) functions as a double-switch gene in the final step of the sex-determination cascade in the silkworm Bombyx mori. The P-element somatic inhibitor (PSI) protein in B. mori interacts with Bmdsx pre-mRNA in CE1 as an exonic splicing silencer to promote male-specific splicing of Bmdsx. However, the character of the interaction between BmPSI and Bmdsx pre-mRNA remains unclear. Electrophoretic mobility shift assay (EMSA) results showed that the four KH_1 motifs in BmPSI are all essential for the binding, especially the former two KH_1 motifs. Three active sites (I116, L127, and IGGI) in the KH_1 motif were found to be necessary for the binding through EMSA, circular dichroism (CD) spectroscopy, and isothermal titration calorimetry (ITC). The PSI homologous protein in S. litura (SlPSI) was purified and the binding of SlPSI and CE1 was verified. Compared with BmPSI, the mutant SlPSI proteins of I116 and IGGI lost their ability to bind to CE1. In conclusion, the binding of PSI and dsx pre-mRNA are generally conserved in both B. mori and S. litura. These findings provide clues for sex determination in Lepidoptera.
Doublesex
Spodoptera litura
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Doublesex
Bombyx
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Bmdsx is an orthologue of the sex-determining gene doublesex(dsx) and known to be sex-specifically expressed in various tissues of the silkworm, Bombyx mori. Its pre-mRNA is sex-specifically spliced and encodes female-specific or male-specific polypeptides. The open reading frame of Bmdsx consists of 5 exons, of which exons 3 and 4 are female-specific and its pre-mRNA was known to undergo default processing to generate the female-type mRNA. Previous reports have shown that the mechanism of splicing of the doublesex gene is different in Drosophila melanogaster and Bombyx mori. However, intron 4 is so long that it is difficult to identify the intronic cis-element(s) required for male-specific splicing of Bmdsx pre-mRNA using Bmdsx minigenes whose introns are shortened in various manners. As a first step toward discovery of the cis-element, the Bmdsx mini gene, which consisted of exon 1 and 5 and internally shortened introns 2 to 4, was constructed, and transgenic silkworms expressing this construct were generated. Bmdsx pre-mRNA transcribed derived from transgene was sex-specifically spliced. This result shows that the mini gene contained the information necessary for the correct regulation of alternative splicing.
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Spodoptera litura
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Piperonyl butoxide
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In Drosophila melanogaster, transformer-2 (tra-2) is essential for female differentiation and is known to induce female-specific splicing of doublesex (dsx). The function of Bmtra-2, the Bombyx mori homolog of tra-2, on the other hand remains to be elucidated. As an initial step to learn about the biological function of Bmtra-2, we determined whether Bmtra-2 is capable of inducing the female-specific splicing of Drosophila dsx. RNAi-mediated knockdown of Bmtra-2 using Bombyx cultured cells transiently transfected with a dsx minigene revealed that Bmtra-2 can induce female-specific splicing of Drosophila dsx. To examine the role Bmtra-2 plays in regulating sex-specific splicing of Bmdsx pre-mRNA, we used an RNAi approach to reduce BmTra-2 expression in the early embryo. Embryos injected with dsRNAs or siRNAs targeted to Bmtra-2 showed no variation in the sex-specific splicing pattern of Bmdsx pre-mRNA. RNAi knockdown of Bmtra-2 in the early embryo caused abnormal testis formation. Taken together, these results indicate that Bmtra-2 is required for normal testis development, but is not involved in regulating the sex-specific splicing of Bmdsx pre-mRNA, even though it is capable of inducing the female-specific splicing of Drosophila dsx.
Doublesex
Minigene
SR protein
Bombyx
Blastoderm
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Doublesex is highly conserved and sex-specifically spliced in insect sex-determination pathways, and its alternative splicing (AS) is regulated by Transformer, an exonic splicing activator, in the model system of Drosophila melanogaster. However, due to the lack of a transformer gene, AS regulation of doublesex remains unclear in Lepidoptera, which contain the economically important silkworm Bombyx mori and thousands of agricultural pests. Here, we use yeast three-hybrid system to screen for RNA-binding proteins that recognize sex-specific exons 3 and 4 of silkworm doublesex (Bm-dsx); this approach identified BxRBP1/Lark binding to the exon 3, and BxRBP2/TBPH and BxRBP3/Aret binding to the exon 4. Investigation of tissues shows that BxRBP1 and BxRBP2 have no sex specificity, but BxRBP3 has – three of its four isoforms are expressed with a sex-bias. Using novel sex-specific silkworm cell lines, we find that BxRBP1 and BxRBP3 directly interact with each other, and cooperatively function as splicing repressors. Over-expression of BxRBP1 and BxRBP3 isoforms efficiently inhibits splicing of the exons 3 and 4 in the female-specific cells and generates the male-specific isoform of Bm-dsx. We also demonstrate that the sex-determination upstream gene Masc regulates alternatively transcribed BxRBP3 isoforms. Thus, we identify a new regulatory mechanism of doublesex AS in the silkworm, revealing an evolutionary divergence in insect sex-determination.
Doublesex
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Doublesex
Melanogaster
Sexual Differentiation
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