Research report
Alternative splicing and promoter use in the human GABRA2 gene

https://doi.org/10.1016/j.molbrainres.2005.03.001Get rights and content

Abstract

GABAA receptors mediate the majority of the fast synaptic inhibition in the mammalian brain. They are the targets of several important drugs, including benzodiazepines, which are used as anxiolytics, sedatives, anti-convulsants, and in the treatment of alcohol withdrawal symptoms. Non-coding variations in GABRA2, the gene encoding the α2 subunit, are associated with the risk for alcoholism, suggesting that regulatory differences are important. GABRA2 mRNAs from whole human brain and from three brain regions were examined for evidence of alternative splicing using reverse transcription-PCR and DNA sequencing. A complex pattern of alternative splicing and alternative promoter use of the human GABRA2 mRNA was demonstrated. There are four major isoforms consisting of combinations of two alternative 5′ and 3′ exons, as well as minor isoforms lacking exon 4 or exon 8. The alternative 5′ exons each lie downstream of a functional promoter sequence, as shown by transient transfection assays. The promoter activities of naturally occurring haplotypes differed, indicating genetic differences in gene expression.

Introduction

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain, with the majority of fast synaptic inhibition mediated through GABAA receptors [1], [2], [27], [46]. GABAA receptors are ligand-gated chloride ion channels [1]. They are pentameric assemblies of subunits from several classes [1], [2], [27], [36], [37], [40]. A variety of important drugs, including benzodiazepines, barbiturates, steroids, anesthetics, and convulsants, act at GABAA receptors [1]. GABAA receptors containing α1, α2, α3, and α5 subunits are targets of benzodiazepines, which are used as anxiolytics, anticonvulsants, sedative-hypnotics, muscle relaxants, and in the treatment of alcohol withdrawal symptoms [1], [14], [25], [36], [37], [43], [46]. The anxiolytic activity of diazepam is mediated by GABAA receptors containing α2 subunits; mice in which a point mutation (H101R) was introduced into the gene encoding the α2 subunit (Gabra2) were insensitive to the anxiolytic effects of diazepam, but retained sensitivity to its sedative and amnestic properties [25].

GABAA receptors are sensitive to ethanol and mediate many of its important effects, including alcohol preference, anxiolysis, sedation, tolerance, motor incoordination, withdrawal, and dependence [4], [11], [14], [15], [22], [45], [46]. In rats, GABAA agonists increase ethanol intake and antagonists decrease ethanol intake [3], [32], [44]. Linkage disequilibrium studies in families with a high density of alcoholics showed that variations in GABRA2 are very strongly associated with the risk for alcoholism [7]. This finding has been replicated in an independent population [6]. Variations in GABRA2 are also associated with electroencephalographic differences in the beta frequency range [7], [34]. However, sequencing the coding regions from more than 20 individuals with each of the two major haplotypes (high- and low-risk) detected no coding difference [7]. This suggested that regulatory differences might affect these phenotypes and prompted an examination of potential alternative splicing and alternative promoter use in the human GABRA2 gene. The homologous rat GABAA receptor α2-subunit gene (Gabra2) has three alternative exons (exons 1A, 1B, and 1C) in the 5′ untranslated region and has been shown to have three alternate promoters [9].

The reference sequence for human GABRA2 mRNA (NM_000807) has 10 exons. We found a second cDNA with different 5′ and 3′ sequences that shares most of the coding region, suggesting alternative splicing and potentially alternative promoter use. Here, we demonstrate the presence of alternative 5′ and 3′ exons as well as alternative use of two internal exons in the human GABRA2 gene, all forms of which were found in several brain regions. Because there were alternative non-coding 5′ exons, the possibility of alternate promoter use was examined using transient transfection assays. The fact that non-coding variations in GABRA2 affect both an electrophysiological phenotype and the risk for alcoholism led us to examine the effect of different haplotypes on promoter function.

Section snippets

Bioinformatics

To identify related sequences, the nr, EST, and human genome sequence databases (NCBI) were searched using the BLAST program, with the RefSeq entry corresponding to the GABRA2 mRNA (NM_000807.1, based upon GenBank S62907) as query. Sequences so identified were further analyzed by BLAST, and predicted proteins were examined using MacVector 7.2.2 (Accelrys, San Diego, CA). Sequence information from NT_006238.10 (6096911 to 5951672) was used to design primers for PCR and sequencing; numbering of

Alternative 5′ and 3′ exons

The prototype for GABRA2 (RefSeq NM_000807.1) was GenBank sequence S62907 [Homo sapiens gamma-aminobutyric acid (GABA) A receptor, alpha 2 (GABRA2), mRNA [13]]. Searching GenBank using the BLAST algorithm revealed that BC022488 [Homo sapiens, clone MGC: 26342, IMAGE: 4812001, mRNA, complete cds] had in common exons 2–9, containing a common coding sequence through much of exon 9 but differed at both 5′ and 3′ ends. Because such differences could be due to cloning artifacts such as chimeric RNAs,

Discussion

This study was prompted by the finding that non-coding variations in the GABRA2 gene were strongly associated with the risk for alcoholism [7] and with electroencephalographic differences in the beta frequency range [7], [34]. The initial association findings were replicated in a second population [6]. This genetic finding makes great biological sense for several reasons. GABA and GABAA receptors are the major mediators of fast synaptic inhibition [1], [27], [46]. Alcohol and many other drugs

Acknowledgments

The authors thank Dr. Joyce Hurley for helpful comments on the manuscript. This study was partially supported by NIH Grants AA06460 and U10AA08403 (the Collaborative Study on the Genetics of Alcoholism) and by the Center for Medical Genomics at Indiana University School of Medicine, which is supported in part by grants from the Indiana 21st Century Research and Technology Fund (HJE) and the Indiana Genomics Initiative (supported in part by the Lilly Endowment).

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