Bmdsx is a sex-determining gene in the silkworm and is alternatively spliced in males and females. CE1 is a splicing silencer element responsible for the sex-specific splicing of Bmdsx. To identify sex-specific factors implicated in the sex-specific splicing of Bmdsx, we performed RNA affinity chromatography using CE1 RNA as a ligand. We have identified BmIMP, a Bombyx homolog of IGF-II mRNA binding protein (IMP), as a male-specific factor that specifically binds to CE1. The gene encoding BmIMP is localized on the Z chromosome and is male-specifically expressed in various tissues. Antisense inhibition of BmIMP expression increased female-specific splicing of Bmdsx pre-mRNA. Coimmunoprecipitation and glutathione S-transferase (GST) pulldown analyses demonstrated that BmIMP physically interacts with BmPSI, which has been identified as a factor implicated in the sex-specific splicing of Bmdsx, through the KH domains of BmIMP. The functional consequence of this interaction was examined using RNA mobility shift analysis. BmIMP increased BmPSI-CE1 RNA binding activity by decreasing the rate of BmPSI dissociation from CE1 RNA. Truncation analysis of BmIMP suggested that the KH domains are responsible for enhancing BmPSI-CE1 RNA binding activity. These results suggest that BmIMP may enhance the male-specific splicing of Bmdsx pre-mRNA by increasing RNA binding activity of BmPSI.Alternative splicing of pre-mRNA is an essential mechanism for differential gene expression that can produce functionally distinct proteins from a single gene according to the developmental or physiological state of cells in multicellular organisms (4,17). Recent studies estimate that about 70% of human genes are alternatively spliced (29), most strikingly, causing several thousands of different mRNA isoforms from a single gene. Although many examples describe how alternative splicing regulates gene expression, the mechanisms involved in the alternative splicing are less well understood (5,9,13,27,28,51). One of the best-studied systems in which the functions of splicing factors are known derives from the Drosophila melanogaster sex determination cascade. At the top of this cascade is the Sex-lethal (Sxl) gene. The functional protein of this master switch gene is produced early in development according to the primary sex determination signal, the X chromosome-to-autosome ratio (X:A ratio) (10,12,14,21). When the functional Sxl protein is produced, it directs the female-specific splicing of its downstream gene transformer (tra), giving rise to functional Tra protein (6). Tra, together with Tra2, binds to the cisregulatory element (dsxRE) within the female-specific exon of its downstream gene doublesex (dsx), activating a weak 3Ј splicing site preceding the female-specific exon to generate the female-type Dsx protein (Dsx F ), which regulates its downstream genes for female development. In males, the X:A ratio of 0.5 inhibits Sxl from being produced, causing male-specific splicing of tra, making its encoded product nonfunctional due to a premature ...