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

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