Analysis of exonic splicing enhancers in the mouse gonadotropin-releasing hormone (GnRH) gene

We have previously reported that efficient removal of the first intron (int ron A) of gonadotropin-releasing hormone (GnRH) pre-mRNA is the prerequisit e event for post-transcriptional regulation. In the present study. using an in vitro HeLa splicing system, we examined the enhancing activities of exo nic elements for GnRH pre-mRNA splicing. While not excised by exon 2 alone, intron A was efficiently excised when exon 3 and/or exon 4 was combined wi th exon 2, suggesting the presence of exonic splicing enhancers (ESEs) in e xons 3 and 4. Purine-rich sequences located in the border of exons 2 and 3 (denoted ESE3) and in exon 4 (ESE4) revealed strong splicing enhancing acti vities. Mutation in ESE3 decreased pre-mRNA splicing, while mutation in pur ine-rich sequences in tron 2 did not. We further analyzed the functional ac tivity of ESE4 by mutations or deletions of the ESE4 sequence that consists of three purine-repeats separated by two spacers and a putative hairpin co nstructing sequence. An UV cross-linking assay using the RNA sequence of ES E4 examined the presence of ESE4-specific binding proteins in the nuclear e xtracts From GT1 hypothalamic GnRH neurons. Collectively, this study indica tes that a sequence context of ESE4 and its binding proteins may be crucial ly involved in enhanced GnRH pre-mRNA splicing. However. it should be furth er clarified as to which splicing factor(s) is responsible for ESE4-depende nt GnRH pre-mRNA splicing. (C) 2001 Elsevier Science Ireland Ltd. All right s reserved.