A Novel Gonadotropin-Releasing Hormone 1 (Gnrh1) Enhancer-Derived Noncoding RNA in the Regulation of Gnrh1 Gene Expression

Gonadotropin-releasing hormone (GnRH), a neuropeptide released from a small population of neurons in the hypothalamus, is the central mediator of the hypothalamic-pituitary-gonadal axis, and is required for normal reproductive development and function. Evolutionarily conserved regulatory elements in the mouse, rat, and human Gnrh1 gene include three enhancers and the proximal promoter, which confer Gnrh1 gene expression specifically in GnRH neurons. In immortalized mouse hypothalamic GnRH (GT1-7) neurons, which show pulsatile release of GnRH in culture, RNA sequencing and RT-qPCR revealed that expression of a novel long noncoding RNA at Gnrh1 enhancer 1 correlates with high levels of Gnrh1 mRNA expression. In GT1-7 neurons, which contain a transgene carrying 3 kb of the rat Gnrh1 regulatory region, both the mouse and rat GnRH-E1 RNAs are expressed. I investigated the characteristics and function of the mouse Gnrh1 enhancer-derived noncoding RNA (GnRH-E1 RNA). Strand-specific RT-PCR analysis of GnRH-E1 RNA in GT1-7 cells revealed sense and antisense GnRH-E1 RNAs that are transcribed from distinct 5’ start sites, are 3’ polyadenylated, and are over 2 kb in length. GnRH-E1 RNAs are long intergenic noncoding RNAs localized in the nucleus with a half-life of 8 hours. In GT1-7 neurons, siRNA knockdown of mouse GnRH-E1 RNA resulted in a significant decrease in the expression of the Gnrh1 primary transcript and Gnrh1 mRNA. Over-expression of either the mouse or rat GnRH-E1 RNA in GT1-7 cells caused a decrease in the transcriptional activity of co-transfected rat Gnrh1 regulatory elements, which suggests a critical role of balanced GnRH-E1 RNA abundance. The action of GnRH-E1 RNA appears to require critical locations in the Gnrh1 regulatory region. However, the over-expression of either the sense or antisense variants of the mouse GnRH-E1 RNA in immature, migratory GnRH (GN11) neurons, which do not express GnRH-E1 RNA or Gnrh1 mRNA, induced the transcriptional activity of co-transfected rat Gnrh1 gene regulatory elements, where the induction requires the presence of the rat Gnrh1 promoter. Together, these data indicate that GnRH-E1 RNA functions as an inducer of Gnrh1 gene transcriptional activity. GnRH-E1 RNA may play an important role in the development and maturation of GnRH neurons