Dynamic regulation of glutamate decarboxylase 67 gene expression by alternative promoters and splicing during rat testis maturation

Abstract

Glutamate decarboxylase produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. Two homologous forms of GAD encoded by separate genes have been identified in mammalian brain, with molecular weight of 67 kDa (GAD67) and 65 kDa (GAD65). Here, we studied the transcriptional regulation of GAD67. Three transcript variants (GAD67A, GAD67B, and GAD67C) transcribed from distinct categories of transcriptional start sites were identified. RT-PCR revealed these transcripts have distinct tissues distributions. Though GAD67A and GAD67B were co-expressed in brain and many nonneural tissues, in heart, only GAD67A was expressed. GAD67C was specifically expressed in testis. These transcripts also showed distinct developmental expression patterns during testis maturation. GAD67A was expressed at all age points examined. GAD67B was only detected at postnatal day 1 and day 5, while GAD67C was expressed from postnatal day 30. Characterizing the genome sequence upstream of transcriptional start sites of these transcripts revealed the presence of TATA-less promoters. Potential promoter activities were analyzed by coupling these promoter sequences to the open reading frame of a luciferase reporter gene in transient expression experiments. Moreover, our results showed GAD67 gene expression was also regulated by alternative splicing in postnatal day 1 and day 5 testis. The above results suggested GAD67 gene expression was dynamically regulated by alternative promoters and splicing during postnatal rat testis maturation.

Removal Kinetics of Antibodies Against Glutamic Acid Decarboxylase by Various Plasmapheresis Modalities in the Treatment of Neurological Disorders

“…Two homologous forms of GAD encoded by separate genes have been identified in the mammalian brain, each with a molecular weight of 65 kDa (GAD 65) and 67 kDa (GAD 67) . Anti‐GAD were measured as antibodies against human recombinant GAD65 by radioimmunoassay test (Cosmic Corp., Tokyo, Japan).…”

Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain

“…The functional significance of these GAD25 proteins in normal embryonic and adult brains, and in pathophysiological conditions is still unclear. It might be implied that the generation of these transcripts is more likely to regulate GAD1 gene expression and the level of GAD activity [ 18 , 24 , 25 , 33 ].…”

“…Human alternatively spliced mRNA is shorter than the GAD1 mRNA and comprises only of the first seven exons of the human GAD1 gene, thus encoding a shorter 25-kDa protein [ 22 , 23 ]. In rat testis and other tissues four novel GAD1 mRNA isoforms synthetized by alternative splicing combined with the utilization of an intron located polyadenylation site and additional transcription start site located in Intron3 were recently described [ 24 , 25 ].…”

Genomic features of BRDT binding sites in gene units suggest transcriptional partners and specific epigenetic landscapes to regulate transcriptional activity during spermatogenesis

“…As we designated the midpoint of the peak as the BRDT binding site, some peaks might have been counted as being present in the first exon even though they partially overlap the promoter as well. Lastly, alternative promoter usage is prevalent in the testis (DeJong, 2006;Freiman, 2009;Liu, Zhang, Li, Yan, & Li, 2010) and some early intronic/exonic binding could in fact be binding of an alternative promoter. Interestingly, 791 of the 1989 (40%) intronic sites of BRDT binding in pachytene spermatocytes and 801 of the 2392 (33%) intronic sites of BRDT binding in round spermatids occur in the first intron ( Figure 1B), the most common location for IME binding.…”