Alternative splicing regulation of doublesexgene by RNA-binding proteins in the silkworm Bombyx mori

ABSTRACTDoublesexis 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 transformergene, AS regulation of doublesexremains unclear in Lepidoptera, which contain the economically important silkworm Bombyx moriand 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 BxRBP1and BxRBP2have no sex specificity, but BxRBP3has – 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 Mascregulates alternatively transcribed BxRBP3 isoforms. Thus, we identify a new regulatory mechanism of doublesexAS in the silkworm, revealing an evolutionary divergence in insect sex-determination.