The Masc–PSI complex directly induces male-type doublesex splicing in silkworms

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Abstract
The WZ sex determination system is found in a diverse range of animals, including lepidopteran insects. In the silkworm Bombyx mori , a lepidopteran model insect, females carry the W chromosome with the feminizing gene Feminizer ( Fem ), which is the source of a female-specific PIWI-interacting RNA (piRNA). The Fem piRNA–PIWI complex cleaves an mRNA encoding the masculinizing protein Masculinizer (BmMasc), resulting in the production of the female-type splice variant of Bombyx mori doublesex ( Bmdsx ), which is the master genetic switch of B. mori sex differentiation. In contrast, in males, BmMasc induces the production of the male-type Bmdsx splice variant ( Bmdsx M ). However, the molecular mechanism through which BmMasc transduces the masculinizing signal to the male-specific Bmdsx splicing event remains unknown. In this study, we showed that BmMasc physically interacts with Bombyx mori P-element somatic inhibitor (BmPSI), that is a RNA binding protein required for Bmdsx M expression. BmMasc overexpression resulted in the production of Bmdsx M in B. mori ovary-derived BmN-4 cells, but this induction was severely inhibited in BmPSI -knocked down cells, indicating that BmPSI is essential for the masculinizing activity of BmMasc. We also identified that BmMasc-containing protein complex was associated with a region of Bmdsx pre-mRNA spanning introns 2 to 4, particularly around the junctions of intron 2 with exon 3 and intron 3 with exon 4. Exons 3 and 4 of Bmdsx are female-specific units and are skipped in Bmdsx M . Together with a previous report of the binding of BmPSI to the CE1 sequence located in exon 4 of Bmdsx , the current results strongly suggest that the BmMasc–BmPSI complex is required for male-specific exon skipping in Bmdsx pre-mRNA through its binding to the female-specific Bmdsx introns and exons.
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