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Submitted on November 14, 2001
Accepted on July 22, 2002
, a 40 kDa Serine/Arginine-Rich (SR)
Protein
1 Hormone Research Center, Chonnam National University, Kwangju 500
757, Korea School of Biological Sciences, Seoul National University, Seoul 151742, Korea Department of Obstetrics and Gynecology, University of Göttingen, Göttingen 37075, Germany * These authors contributed equally to this work.
* To whom correspondence should be addressed. E-mail: kyungjin{at}snu.ac.kr.
In an earlier study, we found that excision of the first intron (intron A) from the rat GnRH (GnRH) primary transcript is attenuated in non-GnRH-producing cells. This attenuation can be partially relieved by exonic splicing enhancers (ESEs) located in GnRH exons 3 and 4. In the present study we confirmed that intron A of the mouse GnRH pre-mRNA was not excised in a HeLa nuclear extract (NE) in vitro or in COS-7 cells in vivo. Intron A could, however, be partially removed when exon 3 and/or 4 were linked to exon 2. In the presence of an ESE in exon 4 (ESE4), an addition of GT1 NE further increased the excision rate of intron A, while the addition of KK1 (a non-GnRH-producing cell) NE decreased it. To define the GnRH neuron-specific splicing activity, GT1 NE was fractionated by ultracentrifugation and ammonium sulfate precipitation. A 5090% ammonium sulfate pellet (ASP5090) fraction was further precipitated with 20 mM MgCl2 to isolate a serine/arginine (SR) protein-rich fraction. Among the ASP fractions, ASP4050 significantly increased the excision rate of intron A in the presence of HeLa NE or SR protein-rich fraction. However, the ASP4050 fraction alone could not remove intron A. This result suggests the presence of a cofactor protein(s) in the ASP4050 fraction that may mediate the interaction between a 3' spliceosome complex and the ESE4-SR protein complex. UV cross-linking and gel mobility shift analysis revealed that Tra2 but not other SR proteins tested, specifically binds to ESE4. Moreover, Tra2 stimulated intron A excision in a dose-dependent manner. These results imply that Tra2 and a cofactor protein in the ASP4050 fraction are involved in mediating the GnRH neuron-specific excision of intron A from the GnRH primary transcript.
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