A Novel Splice Variant of the Masculinizing Gene <i>Masc</i> with piRNA-Cleavage-Site Defect Functions in Female External Genital Development in the Silkworm, <i>Bombyx mori</i>

• Main Authors: Qin Zhao, Juan Li, Mao-Yu Wen, He Wang, Yao Wang, Kai-Xuan Wang, Qiu-Xing Wan, Xing-Fu Zha
• Format: Article
• Language: English
• Published: MDPI AG 2019-07-01
• Series: Biomolecules
• Subjects: silkworm; sex determination; alternative splicing; <i>Masc</i>; BmEXU; <i>Adb-B</i>
• Online Access: https://www.mdpi.com/2218-273X/9/8/318

In the silkworm, the sex-determination primary signal <i>Fem</i> controls sex differentiation by specific binding of <i>Fem</i>-derived piRNA to the cleavage site in <i>Masc</i> mRNA, thus inhibiting <i>Masc</i> protein production in the female. In this study, we identified a novel splicing isoform of <i>Masc</i>, named <i>Masc-S</i>, which lacks the intact sequence of the cleavage site, encoding a C-terminal truncated protein. Results of RT-PCR showed that <i>Masc-S</i> was expressed in both sexes. Over-expression of <i>Masc-S</i> and <i>Masc</i> in female-specific cell lines showed that <i>Masc-S</i> could be translated against the <i>Fem</i>-piRNA cut. By RNA-protein pull-down, LC/MS/MS, and EMSA, we identified a protein BmEXU that specifically binds to an exclusive RNA sequence in <i>Masc</i> compared to <i>Masc-S</i>. Knockdown of <i>Masc-S</i> resulted in abnormal morphology in female external genital and increased expression of the Hox gene <i>Abd-B</i>, which similarly occurred by <i>Bmexu</i> RNAi. These results suggest that the splice variant <i>Masc-S</i> against <i>Fem</i>-piRNA plays an important role in female external genital development, of which function is opposite to that of full-length <i>Masc</i>. Our study provides new insights into the regulatory mechanism of sex determination in the silkworm.