The intronic GABRG2 mutation, IVS6+2T→G, associated with childhood absence epilepsy altered subunit mRNA intron splicing, activated nonsense-mediated decay, and produced a stable truncated γ2 subunit

Mengnan Tian, Robert L. Macdonald

Research output: Contribution to journalArticle

Abstract

The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wild-type and mutant γ2 subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wild-type and mutant γ2 subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wild-type and mutant GABRG2 mRNA splicing patterns were determined in both BAC-transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention, and produced a frameshift in exon 7 that created a premature translation termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense-mediated mRNA decay or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first six GABRG2 exons and a novel frameshifted 29 aa C-terminal tail. The γ2-PTC subunit was homologous to the mollusk AChBP (acetylcholine binding protein) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant-negative effect on αβγ 2 receptor assembly.

Original languageEnglish (US)
Pages (from-to)5937-5952
Number of pages16
JournalJournal of Neuroscience
Volume32
Issue number17
DOIs
StatePublished - Apr 25 2012
Externally publishedYes

Fingerprint

Bacterial Artificial Chromosomes
Absence Epilepsy
Nonsense Codon
Introns
RNA Splice Sites
Messenger RNA
Mutation
Transgenic Mice
Exons
Nonsense Mediated mRNA Decay
Febrile Seizures
Mollusca
Acetylcholine
Tail
Carrier Proteins
Membranes
Brain

ASJC Scopus subject areas

  • Neuroscience(all)

Cite this

@article{7ad6be8fbea2429dafb2230e78d61450,
title = "The intronic GABRG2 mutation, IVS6+2T→G, associated with childhood absence epilepsy altered subunit mRNA intron splicing, activated nonsense-mediated decay, and produced a stable truncated γ2 subunit",
abstract = "The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wild-type and mutant γ2 subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wild-type and mutant γ2 subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wild-type and mutant GABRG2 mRNA splicing patterns were determined in both BAC-transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention, and produced a frameshift in exon 7 that created a premature translation termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense-mediated mRNA decay or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first six GABRG2 exons and a novel frameshifted 29 aa C-terminal tail. The γ2-PTC subunit was homologous to the mollusk AChBP (acetylcholine binding protein) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant-negative effect on αβγ 2 receptor assembly.",
author = "Mengnan Tian and Macdonald, {Robert L.}",
year = "2012",
month = "4",
day = "25",
doi = "10.1523/JNEUROSCI.5332-11.2012",
language = "English (US)",
volume = "32",
pages = "5937--5952",
journal = "Journal of Neuroscience",
issn = "0270-6474",
publisher = "Society for Neuroscience",
number = "17",

}

TY - JOUR

T1 - The intronic GABRG2 mutation, IVS6+2T→G, associated with childhood absence epilepsy altered subunit mRNA intron splicing, activated nonsense-mediated decay, and produced a stable truncated γ2 subunit

AU - Tian, Mengnan

AU - Macdonald, Robert L.

PY - 2012/4/25

Y1 - 2012/4/25

N2 - The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wild-type and mutant γ2 subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wild-type and mutant γ2 subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wild-type and mutant GABRG2 mRNA splicing patterns were determined in both BAC-transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention, and produced a frameshift in exon 7 that created a premature translation termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense-mediated mRNA decay or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first six GABRG2 exons and a novel frameshifted 29 aa C-terminal tail. The γ2-PTC subunit was homologous to the mollusk AChBP (acetylcholine binding protein) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant-negative effect on αβγ 2 receptor assembly.

AB - The intronic GABRG2 mutation, IVS6+2T→G, was identified in an Australian family with childhood absence epilepsy and febrile seizures (Kananura et al., 2002). The GABRG2 intron 6 splice donor site was found to be mutated from GT to GG. We generated wild-type and mutant γ2 subunit bacterial artificial chromosomes (BACs) driven by a CMV promoter and expressed them in HEK293T cells and expressed wild-type and mutant γ2 subunit BACs containing the endogenous hGABRG2 promoter in transgenic mice. Wild-type and mutant GABRG2 mRNA splicing patterns were determined in both BAC-transfected HEK293T cells and transgenic mouse brain, and in both, the mutation abolished intron 6 splicing at the donor site, activated a cryptic splice site, generated partial intron 6 retention, and produced a frameshift in exon 7 that created a premature translation termination codon (PTC). The resultant mutant mRNA was either degraded partially by nonsense-mediated mRNA decay or translated to a stable, truncated subunit (the γ2-PTC subunit) containing the first six GABRG2 exons and a novel frameshifted 29 aa C-terminal tail. The γ2-PTC subunit was homologous to the mollusk AChBP (acetylcholine binding protein) but was not secreted from cells. It was retained in the ER and not expressed on the surface membrane, but it did oligomerize with α1 and β2 subunits. These results suggested that the GABRG2 mutation, IVS6+2T→G, reduced surface αβγ2 receptor levels, thus reducing GABAergic inhibition, by reducing GABRG2 transcript level and producing a stable, nonfunctional truncated subunit that had a dominant-negative effect on αβγ 2 receptor assembly.

UR - http://www.scopus.com/inward/record.url?scp=84860200746&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84860200746&partnerID=8YFLogxK

U2 - 10.1523/JNEUROSCI.5332-11.2012

DO - 10.1523/JNEUROSCI.5332-11.2012

M3 - Article

C2 - 22539854

AN - SCOPUS:84860200746

VL - 32

SP - 5937

EP - 5952

JO - Journal of Neuroscience

JF - Journal of Neuroscience

SN - 0270-6474

IS - 17

ER -