GnRH RNA Splicing : (Its Molecular Regulation and Functional Relevance)

다국어 초록 (Multilingual Abstract)

Hypothalamic GnRH is a pivotal regulator of neuroendocrine network in the mammalian reproduction and sexual development. We recently found that excision of the first intron(intron A) of the GnRH pre-mRNA was attenuated in non-GnRH producing cells, which can be partially relieved by exonic splicing enhancers(ESEs) located in GnRH exons 3 and 4(Mol. Endocrinol. 1999, 13:1882). We extended this topic by addressing several other issues on molecular regulation and physiological significance of the GnRH pre-mRNA splicing: 1) To elucidate the functional importance of the ESEs in vivo, we examined the splicing activity of the GnRH pre-mRNA in hypogonadal(hpg) mice of which GnRH exons 3 and 4 are truncated. Intron A excision rate in the preoptic area of hpg mice was severely lower than that of normal mice. As revealed by GnRH-luciferase fusion constructs, intron A-containing GnRH transcript cannot be translated, although GnRH coding sequence starts at the second exon. Thus, failure in removing intron A in hpg GnRH transcripts may contribute to the complete lack of GnRH peptide in these mutant mice. 2) Since in the presence of ESEs, an addition of GT1 nuclear extract further increased excision rate of intron A, we attempted to define the possible GnRH neuron-specific trans-acting factor(s) interacting with ESEs. GT1 nuclear extract was fractionated by ammonium sulfate precipitation(ASP) procedures. Among ASP fractions, ASP40-50 fraction strongly increased the excision of intron A. Moreover, UV cross-linking assay demonstrated that about 40kDa protein(s) in SR protein-rich fraction binds to ESE4. 3) To further define which protein(s) recognize the ESEs, several SR proteins including Tra2. SRp30c, 9G8, SRp55, SRp40 and SRp20 were purified using the baculovirus expression system. Among these SR proteins, Tra2 specifically bound to ESE4, and SRp30c bound to ESE3. Furthermore, yeast two-hybrid screening and GST pull-down assay showed interaction between Tra2 and SRp30c. In contrast to Tra2 and SRp30c, which could not enhance intron A excision in the absence of other nuclear components, 9G8 efficiently removed the GnRH intron A only in the presence of cytoplasmic S100 supplement. These results suggest that excision rate of GnRH intron A can be achieved by protein-RNA complexes including Tra2, SRp30c and 9G8. Therefore, the identification of GnRH neuron-specific splicing machinery responsible for cell type- and developmental stage-specific removal of intron A will provide a novel insight into the molecular regulation of GnRH gene expression.