The Masc-PSI complex directly induces male-type doublesex splicing in silkworms
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 (BmdsxM). 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 BmdsxM expression. BmMasc overexpression resulted in the production of BmdsxM 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 BmdsxM. 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.