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Molecular and Cellular Biology, January 2008, p. 333-343, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01528-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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{triangledown} ,{dagger}

Masataka G. Suzuki,1 Shigeo Imanishi,2 Naoshi Dohmae,3 Tomoe Nishimura,3 Toru Shimada,4 and Shogo Matsumoto1*

Laboratory of Molecular Entomology, The Institute of Physical and Chemical Research 2-1, Hirosawa, Wako, Saitama 351-0198, Japan,1 National Institute of Agrobiological Science, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan,2 Biomolecular Characterization Team, Advanced Development and Supporting Center, The Institute of Physical and Chemical Research 2-1, Hirosawa, Wako, Saitama 351-0198, Japan,3 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan4

Received 21 August 2007/ Returned for modification 27 September 2007/ Accepted 10 October 2007

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.

* Corresponding author. Mailing address: Laboratory of Molecular Entomology, The Institute of Physical and Chemical Research 2-1, Hirosawa, Wako, Saitama 351-0198, Japan. Phone: 81-48-467-9584. Fax: 81-48-462-4678. E-mail: smatsu{at}riken.jp

{triangledown} Published ahead of print on 29 October 2007.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, January 2008, p. 333-343, Vol. 28, No. 1
0270-7306/08/$08.00+0     doi:10.1128/MCB.01528-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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