Analysis of interaction between Bmhrp28 and BmPSI in sex-specific splicing of Bombyx mori Bmdsx gene.

ABSTRACT

Bombyx mori BmHRP28 and BmPSI, which belong to the family of RNA-binding proteins, have been identified binding to the female-specific exon 4 of the sex-determining gene Bmdsx pre-mRNA. However, the relationships between BmHRP28 and BmPSI still remain unclear. In this study, we carried out yeast two-hybrid (Y2H) and co-immunoprecipitation (Co-IP) analyses to address them. Y2H analysis showed that there was little or no direct binding between the BmHRP28 and BmPSI proteins. Also, the Co-IP experiments revealed that BmHRP28 and BmPSI coexisted in a multiprotein complex. Our results suggested that BmHRP28 and BmPSI form a muliprotein complex to regulate the splicing of Bmdsx pre-mRNA, but are not directly bound to each other. In an effort to find other regulatory factors in the multiprotein complex, we constructed a silkworm Y2H cDNA library of male early embryo. By Y2H screening, we identified an RNA-binding protein BmSPX, a putative component of the spliceosome, binding to BmPSI. These results indicated that BmHRP28 and BmPSI make up a spliceosome complex to regulate Bmdsx splicing and that BmSPX is another potential protein involved in this process. Our study provides some clues to better understand the mechanism of sex determination in the silkworm.