Establishment of a Novel In Vivo Sex-Specific Splicing Assay System To Identify a trans-Acting Factor That Negatively Regulates Splicing of Bombyx mori dsx Female Exons

Abstract

The Bombyx mori homolog of doublesex, Bmdsx, plays an essential role in silkworm sexual development. Exons 3 and 4 of Bmdsx pre-mRNA are specifically excluded in males. To explore how this occurs, we developed a novel in vivo sex-specific splicing assay system using sexually differentiated cultured cells. A series of mutation analyses using a Bmdsx minigene with this in vivo splicing assay system identified three distinct sequences (CE1, CE2, and CE3) positioned in exon 4 as exonic splicing silencers responsible for male-specific splicing. Gel shift analysis showed that CE1 binds to a nuclear protein from male cells but not that from female cells. Mutation of UAA repeats within CE1 inhibited the binding of the nuclear protein to the RNA and caused female-specific splicing in male cells. We have identified BmPSI, a Bombyx homolog of P-element somatic inhibitor (PSI), as the nuclear factor that specifically binds CE1. Down-regulation of endogenous BmPSI by RNA interference significantly increased female-specific splicing in male cells. This is the first report of a PSI homolog implicated in the regulated sex-specific splicing of dsx pre-mRNA.Sexual reproduction is the primary means to maintain variation for evolutionary survival. Sex determination cascades that drive the differentiation of dimorphic gonads, however, are some of the most rapidly evolved developmental events (28). Genetic and environmental cues direct the generation of two distinct sexual phenotypes via several key molecular signals. In Drosophila melanogaster and Caenorhabditis elegans, the ratio of X chromosomes to autosomes (X/A) is the primary signal for sex determination (19,23). In mammals, however, maleness is determined by the Y-linked gene SRY (17,41). Nevertheless, recent studies indicate some downstream sex determination genes are functionally similar in diverse species. Of these, DM domain (Doublesex/Mab-3 DNA-binding motif) genes have been shown in the past several years to regulate sexual development in multiple metazoan phyla, including arthropods, nematodes, and vertebrates, and thus this gene family may represent the first example of widespread conservation of sexual regulatory genes (20,46). The DM domain is a cysteine-rich DNA-binding motif first recognized in proteins encoded by the Drosophila sex determination gene doublesex (10, 48). As the name doublesex (dsx) suggests, this gene functions in both sexes: the primary transcript of dsx undergoes sex-specific alternative splicing, producing either a male-specific isoform, DSXM, or a female-specific isoform, DSXF (3, 6). Throughout most of the somatic tissues of the fruit fly, DSXM directs male development and DSXF directs female development.The mechanism of sex-specific dsx splicing has been well studied. Female-specific splicing of dsx requires TRA, TRA-2, and an exonic splicing enhancer (ESE) element located within the untranslated region of the fourth exon (29,34,35). Both TRA and TRA-2 contain Arg/Ser-rich (RS) domains, protein interaction domains characteristic of the Ser/Ar...

Insect sex determination: it all evolves around transformer

“…A tra ortholog has not (yet) been identified in Lepidoptera, perhaps because of the strong sequence divergence that characterizes tra evolution. In Bombyx mori no tra ortholog has been found based on the lack of dsxRE or PRE binding sites on Bmdsx and the presumed default mode of female-specific splicing [43,44]. However, dsxRE/PRE binding sites have only been identified in dipterans based on homology to Drosophila and are probably so diverged that recognition of these sites in other orders is difficult.…”

Effect of RNAi-mediated knockdown of the Bombyx mori transformer-2 gene on the sex-specific splicing of Bmdsx pre-mRNA

“…1 mg of the hsp-dsx minigene was transfected into BmN cells with or without 1 mg of hsp-tra or 1 mg of hsp-tra2 as shown in Fig. 1 according to the protocol described previously (Suzuki et al, 2008).…”

“…We have demonstrated that female splicing of Bmdsx pre-mRNA represents the default mode when tested in HeLa nuclear extracts, and also that the female exon is devoid of putative TRA/TRA-2 binding sites (Suzuki et al, 2001). A splicing inhibitor, BmPSI is involved in the regulation of male-specific splicing of Bmdsx (Suzuki et al, 2008). Since BmPSI does not exhibit any sequence relationship to known SR proteins, such as Tra and Tra-2, the regulatory mechanism of sex-specific alternative splicing of Bmdsx is distinct from that of dsx.…”

An Unusual Role for doublesex in Sex Determination in the Dipteran Sciara

“…Finally, no BmPSI-exonic splicing suppressor (BmPSI-ESS) sequences were detected in Scdsx pre-mRNA. These ESS sequences are found in dsx from the lepidopterans Bombyx and Antheraea (Shukla and Nagaraju 2010), which are targets for the sex-specific factor BmPSI that leads to skipping of the female-specific exons 3 and 4 in males (Suzuki et al 2008).…”

“…Finally, no BmPSI-exonic splicing suppressor (BmPSI-ESS) sequences were detected in Scdsx pre-mRNA. These ESS sequences are found in dsx from the lepidopterans Bombyx and Antheraea (Shukla and Nagaraju 2010), which are targets for the sex-specific factor BmPSI that leads to skipping of the female-specific exons 3 and 4 in males (Suzuki et al 2008).Since the binding sequences of SR proteins, such as TRA and TRA2, may be quite degenerate (Black 2003), and because the DsxRE sequences of Scdsx showed a high degree of similarity with the consensus TRA-TRA2 binding sequences, a possibility existed that a female-specific TRA-TRA2 complex was involved in the quantitative differential splicing of dsx pre-mRNA in Sciara males and females, with that complex favoring the female against the male type. To test this, a Scdsx minigene (DScmg) was constructed that contained all the complete exons E2, E3, and E4 and 276 bp from the 39 end of common exon E1, together with their adjacent intron sequences containing the binding sites surrounding the exons described above (see Materials and Methods and Figure S2 and its legend), and DScmg-transgenic Drosophila flies were produced.…”