Enhanced Splicing of the First Intron from the Gonadotropin-Releasing Hormone (GnRH) Primary Transcript Is a Prerequisite for Mature GnRH Messenger RNA: Presence of GnRH Neuron-Specific Splicing Factors

Abstract

The rat GnRH gene consists of four short exons (denoted 1, 2, 3, and 4) and three introns (A, B, and C). All three introns are spliced from the primary transcript, resulting in a mature mRNA. Northern blot and RT-PCR analyses showed that the GnRH primary transcript and its splicing intermediates are more prevalent than the mature GnRH mRNA in a variety of non-GnRH-producing tissues. To delineate the possible splicing mechanism of introns, an in vitro HeLa splicing system was used. Introns B and C were efficiently spliced, while intron A spanning between exon 1 and exon 2 was not. The retention of intron A was relieved when the 5′- and/or 3′-splice sites of intron A were point mutated based on the consensus sequence. The splicing activity was even more strengthened when a putative branchpoint site was moved to the upstream region of the pyrimidine tract of intron A. Intron A could be partially spliced when whole exons (2, 3, and 4) were linked up with intron A. There are two putative exonic splicing enhancers (ESEs) in exon 3 and exon 4. The ESE on exon 4 (ESE4) is much stronger than that on exon 3. The closer the ESE4 to the 3′-splice site of intron A, the better the splicing activity became. However, in the presence of the nuclear extract from GnRH neurons, there was an enhancement in the splicing activity notwithstanding the distance between ESE4 and 3′-splice site of intron A. These results suggest that the ESE4 functions as both the constitutive and regulated enhancer. Collectively, our study provides evidence that enhanced splicing of intron A by putative GnRH neuron-specific splicing factor(s) interacting with the ESEs is a prerequisite for mature GnRH synthesis.