Cooperative Actions of Tra2α with 9G8 and SRp30c in the RNA Splicing of the Gonadotropin-releasing Hormone Gene Transcript

doi:10.1074/jbc.M505814200

Cooperative Actions of Tra2α with 9G8 and SRp30c in the RNA Splicing of the Gonadotropin-releasing Hormone Gene Transcript^*

^‡School of Biological Sciences, Seoul National University, Seoul 151-742, Korea and the ^§Graduate School of Medicine, Korea University College of Medicine, Seoul 136-705, Korea

3 To whom correspondence should be addressed. Tel.: 82-2-880-6694; Fax: 82-2-884-6560; E-mail: kyungjin{at}snu.ac.kr.

Abstract

In earlier studies, we demonstrated that excision of the first intron (intron A) from the gonadotropin-releasing hormone (GnRH) transcript is highly cell type- and developmental stage-specific. The removal of GnRH intron A requires exonic splicing enhancers on exons 3 and 4 (ESE3 and ESE4, respectively). Tra2α,a serine/arginine-rich (SR)-like protein, specifically binds to ESE4, although it requires additional nuclear co-factors for efficient removal of this intron. In the present study, we demonstrate the cooperative action of multiple SR proteins in the regulation of GnRH pre-mRNA splicing. SRp30c specifically binds to both ESE3 and ESE4, whereas 9G8 binds to an element in exon 3 and strongly enhances the excision of GnRH intron A in the presence of minimal amount of other nuclear components. Interestingly, Tra2α can interact with either 9G8 or SRp30c, whereas no interaction between 9G8 and SRp30c is observed. Tra2α has an additive effect on the RNA binding of these proteins. Overexpression or knock-down of these three proteins in cultured cells further suggests their essential role in intron A excision activities, and their presence in GnRH neurons of the mouse preoptic area further strengthens this possibility. Together, these results indicate that interaction of Tra2α with 9G8 and SRp30c appears to be crucial for ESE-dependent GnRH pre-mRNA splicing, allowing efficient generation of mature mRNA in GnRH-producing cells.

Footnotes

↵4 The abbreviations used are: GnRH, gonadotropin-releasing hormone; SR, serine/arginine-rich; ESE, exonic splicing enhancer; POA, preoptic area; NE, nuclear extract; GST, glutathione S-transferase; RT, reverse transcription; EMSA, electrophoretic mobility shift assay(s); BD, binding domain; AS, antisense; ODN, oligodeoxynucleotide(s); CTL, control; PBS, phosphate-buffered saline; GFP, green fluorescent protein.
↵5 E. Park, J. Han, G. H. Son, M. S. Lee, S. Chung, S. H. Park, K. Park, K. H. Lee, S. Choi, J. Y. Seong, and K. Kim, unpublished observation.
↵* This work was supported by a grant from the Brain Research Center of the 21st Century Frontier Research Program funded by the Ministry of Science and Technology, the Republic of Korea. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵ The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.
↵1 These authors contributed equally to this work.
↵2 Supported by a Brain Korea 21 Research Fellowship from the Korea Ministry of Education and Human Resources.
- Received May 27, 2005.
- Revision received October 14, 2005.
The American Society for Biochemistry and Molecular Biology, Inc.