Identification of a Male-Specific RNA Binding Protein That Regulates Sex-Specific Splicing of Bmdsx by Increasing RNA Binding Activity of BmPSI

Abstract

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 ...

Two Conserved Cysteine Residues Are Required for the Masculinizing Activity of the Silkworm Masc Protein

“…The male-type splicing of Bmdsx is regulated by BmIMP (15), so the Masc-induced increase in the BmIMP protein is considered to directly control the splicing of Bmdsx. Thus, our next goal is to unravel the signaling cascade from the Masc protein to BmIMP transcriptional induction.…”

“…Transfection of a B. mori ovary-derived cell line, BmN-4, with Masc cDNA produced the male-type splice variants of Bmdsx (6), as well as enhanced the expression of BmIMP (B. mori IGF-II mRNA-binding protein) (13,14), the product of which is involved in the male-specific splicing of Bmdsx (15). These results indicate that the masculinizing activity of the Masc protein can be assessed in BmN-4 cells by transfecting Masc cDNA derivatives.…”

Sex Determination in Insects: Variations on a Common Theme

“…Instead, Bmdsx appears to be controlled by a male-specific factor, BmIMP, a Bombyx homolog of IGF-II mRNA-binding protein (IMP) [Suzuki et al, 2010]. Because of the wide use of the tra-dsx module in holometabolous insects, we propose that, in the course of Bombyx evolution, the control of dsx by tra may have been replaced by a novel BmIMP-based regulatory system.…”

Lepidopteran Sex Determination: A Cascade of Surprises

“…On this basis, Suzuki et al [2010] hypothesized the existence of a sex-specific cofactor. Another splicing regulator (BmIMP) of Bmdsx pre-mRNA was identified in a search for male-specific binding partner(s) of BmPSI [Suzuki et al, 2010]. BmIMP is a Bombyx homolog of IGF-II mRNA-binding protein (IMP) [Suzuki et al, 2010].…”

“…Further, Bombyx homolog of P-element somatic inhibitor (Bmpsi) and Bombyx homolog of IGF-II mRNA-binding protein (Bmimp) (discussed later) have recently been reported to be involved in the Bombyx sex determination pathway [Suzuki et al, 2008[Suzuki et al, , 2010.…”