Son Gi Hoon, Jung Hosung, Seong Jae Young, Choe Youngshik, Geum Dongho, Kim Kyungjin
School of Biological Sciences, Seoul National University, Seoul 151-742, Korea.
J Biol Chem. 2003 May 16;278(20):18037-44. doi: 10.1074/jbc.M209850200. Epub 2003 Mar 13.
The mammalian gonadotropin-releasing hormone (GnRH) gene consists of four short exons (denoted as 1, 2, 3, and 4) and three intervening introns (A, B, and C). Recently, we demonstrated that excision of the first intron (intron A) from the GnRH transcript is regulated in a tissue- and developmental stage-specific fashion and is severely attenuated in hypogonadal (hpg) mouse because of its lack of exonic splicing enhancers (ESE) 3 and 4. In the present study, we examined the influence of intron A on translational efficiency, thereby establishing a post-transcriptional control over GnRH biosynthesis. First, we verified that an intron A-retained GnRH transcript is a splicing variant but not a splicing intermediate. Intron A-retained transcripts can be transported to the cytoplasm in contrast to intron B-containing transcripts, which are restricted to the nucleus. This result implicates the intron A-retained GnRH transcript as a splicing variant; it has a long 5'-untranslated region, as the GnRH prohormone open reading frame (ORF) begins on exon 2. We investigated whether an intron A-retained GnRH transcript can properly initiate translation at the appropriate start codon and found that intron A completely blocks the translation initiation of its downstream reporter ORF both in vivo and in vitro. The inhibition of translation initiation appears to be due to the presence of a tandem repeat of ATG sequences within intron A. Constructs bearing mutations of ATGs to AAGs restored translation initiation at the downstream start codon; the extent of this restoration correlated with the number of mutated ATGs. Besides the failure in the translation initiation of GnRH-coding region in the intron A-containing variant, the present study also suggests that the interference between mature GnRH mRNA and intron A-retained splicing variant could occur to lower the efficiency of GnRH biosynthesis in the GT1-1-immortalized GnRH-producing cell line. Therefore, our results indicate that the precise and efficient excision of intron A and the joining of adjacent exons may be a critical regulatory step for the post-transcriptional regulation of GnRH biosynthesis.
哺乳动物促性腺激素释放激素（GnRH）基因由四个短外显子（分别标记为1、2、3和4）和三个间隔内含子（A、B和C）组成。最近，我们证明，从GnRH转录本中切除第一个内含子（内含子A）是以组织和发育阶段特异性方式调控的，并且在性腺功能减退（hpg）小鼠中由于缺乏外显子剪接增强子（ESE）3和4而严重减弱。在本研究中，我们研究了内含子A对翻译效率的影响，从而建立了对GnRH生物合成的转录后调控。首先，我们验证了保留内含子A的GnRH转录本是一种剪接变体而非剪接中间体。与局限于细胞核的含内含子B的转录本不同，保留内含子A的转录本能够转运至细胞质。这一结果表明保留内含子A的GnRH转录本是一种剪接变体；它具有长的5'非翻译区，因为GnRH前体激素开放阅读框（ORF）始于外显子2。我们研究了保留内含子A的GnRH转录本是否能在合适的起始密码子处正确起始翻译，结果发现内含子A在体内和体外均完全阻断其下游报告基因ORF的翻译起始。翻译起始的抑制似乎是由于内含子A中存在ATG序列的串联重复。将ATG突变为AAG的构建体恢复了下游起始密码子处的翻译起始；这种恢复程度与突变的ATG数量相关。除了含内含子A的变体中GnRH编码区翻译起始失败外，本研究还表明，在GT1-1永生化GnRH产生细胞系中，成熟GnRH mRNA与保留内含子A的剪接变体之间可能发生干扰，从而降低GnRH生物合成效率。因此，我们的结果表明，内含子A的精确有效切除以及相邻外显子的连接可能是GnRH生物合成转录后调控的关键调节步骤。
