Male-specific splicing of the silkworm Imp gene is maintained by an autoregulatory mechanism

• The terminal exon of Imp is male-specifically included.
• Imp protein is required for the male-specific splicing of its own pre-mRNA.
• Male-specific Imp splicing requires A-rich elements in the terminal intron.
• Recombinant Imp protein specifically binds to A-rich elements.
• Male-specific splicing of Imp is controlled via its autoregulatory actions.

Sexual differentiation in the silkworm Bombyx mori is controlled by sex-specific splicing of Bmdsx, in which exons 3 and 4 are skipped in males. B. mori insulin-like growth factor II mRNA-binding protein (Imp) is a factor involved in the male-specific splicing of Bmdsx. In this study, we found that the male-specific Imp mRNA is formed as a result of the inclusion of exon 8 and the promoter-distal poly(A) site choice, whereas non-sex-specific polyadenylation occurs at the promoter-proximal poly(A) site downstream of exon 7. Recent studies revealed that Drosophila Sxl, tra in several dipteran and hymenopteran insects, and fem in Apis mellifera, play a central role in sex determination and maintain their productive mode of expression via an autoregulatory function. To determine whether Imp protein is required for the maintenance of the male-specific splicing of its own pre-mRNA, we knocked down endogenous Imp in male cells and assessed the male-specific splicing of an exogenous Imp minigene. Knockdown of endogenous Imp inhibited the male-specific splicing of the Imp minigene transcript. In contrast, overexpression of Imp in female cells induced the male-specific splicing of the Imp minigene transcript. Moreover, deletion of adenine-rich (A-rich) sequences located downstream of the proximal poly(A) site repressed the male-specific splicing of the Imp minigene transcript. Finally, gel shift analysis demonstrated that Imp binds to the A-rich sequences. These data suggest that Imp binds to the A-rich sequences in its own pre-mRNA to induce the male-specific splicing of its pre-mRNA.