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.

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[82]  M. Rosbash,et al.  Intramolecular structure in yeast introns aids the early steps of in vitro spliceosome assembly. , 1996, RNA.

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[84]  S. Richard,et al.  p62 Association with RNA Is Regulated by Tyrosine Phosphorylation (*) , 1995, The Journal of Biological Chemistry.

[85]  T. Maniatis,et al.  Synergistic interactions between two distinct elements of a regulated splicing enhancer. , 1995, Genes & development.

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[87]  K. Burtis,et al.  The Drosophila doublesex proteins share a novel zinc finger related DNA binding domain. , 1993, The EMBO journal.

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[92]  D. Rio,et al.  Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression. , 1990, Science.

[93]  B. S. Baker,et al.  Regulation of sex-specific RNA splicing at the Drosophila doublesex gene: cis-acting mutations in exon sequences alter sex-specific RNA splicing patterns. , 1990, Genes & development.

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[96]  B. S. Baker,et al.  The control of alternative splicing at genes regulating sexual differentiation in D. melanogaster , 1988, Cell.

[97]  B. S. Baker,et al.  A molecular analysis of doublesex, a bifunctional gene that controls both male and female sexual differentiation in Drosophila melanogaster. , 1988, Genes & development.