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Exonic Splicing Enhancer-Dependent Splicing of the Gonadotropin-Releasing Hormone Premessenger Ribonucleic Acid Is Mediated by Tra2, a 40-Kilodalton Serine/Arginine-Rich Protein

Hormone Research Center (J.Y.S.), Chonnam National University, Kwangju 500-757, Korea; School of Biological Sciences (J.H., S.P., K.K.), Seoul National University, Seoul 151-742, Korea; and Department of Obstetrics and Gynecology (W.W., H.J.), University of Göttingen, Göttingen 37075, Germany

Address all correspondence and requests for reprints to: Kyungjin Kim, Ph.D., School of Biological Sciences Seoul National University Seoul 151-742, Korea. E-mail: .

In an earlier study, we found that excision of the first intron (intron A) from the rat 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, whereas 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 50–90% ammonium sulfate pellet (ASP50–90) fraction was further precipitated with 20 mM MgCl₂ to isolate a serine/arginine-rich (SR) protein fraction. Among the ASP fractions, ASP40–50 significantly increased the excision rate of intron A in the presence of HeLa NE or SR protein-rich fraction. However, the ASP40–50 fraction alone could not remove intron A. This result suggests the presence of a cofactor protein(s) in the ASP40–50 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 ASP40–50 fraction are involved in mediating the GnRH neuron-specific excision of intron A from the GnRH primary transcript.

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