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.
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.
Suzuki
Masataka G
MG
Laboratory of Molecular Entomology, The Institute of Physical and Chemical Research 2-1, Hirosawa, Wako, Saitama 351-0198, Japan. smatsu@riken.jp
Imanishi
Shigeo
S
Dohmae
Naoshi
N
Nishimura
Tomoe
T
Shimada
Toru
T
Matsumoto
Shogo
S
D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000235:genetics*; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation / Q000235:genetics*; D005091:Exons / Q000235:genetics*; D005260:Female; D005821:Genetic Techniques; D008297:Male; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D012326:RNA Splicing / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012727:Sex Characteristics; D015534:Trans-Activators / Q000235:genetics* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators