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Alternative-splicing in the exon-10 region of GABA(A) receptor beta(2) subunit gene: relationships between novel isoforms and psychotic disorders.

Zhao C, Xu Z, Wang F, Chen J, Ng SK, Wong PW, Yu Z, Pun FW, Ren L, Lo WS, Tsang SY, Xue H - PLoS ONE (2009)

Bottom Line: Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression.Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Applied Genomics Center, Fok Ying Tung Graduate School, The Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT

Background: Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for beta(2)-subunit of gamma-aminobutyric acid type A (GABA(A)) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing.

Methods and findings: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1) and beta(2S2), bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1) expression and decreased beta(2S2) expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression. Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.

Conclusion: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.

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Characterizations of Exon 10 of GABRB2.(A) The exonic structure of human GABRB2 showing positions of the tagging SNPs, and the alignment of its Exon 10 with corresponding sequences in other vertebrates (from UCSC Genome Browser). The lengths of the sequences corresponding to the intervening sequences between E9 and E11 in the different species are indicated on the right. Dots represent identities; single line, deletions; double-dashed line,unalignable bases. Exons are shown as numbered boxes. (B) RT-PCR analyses of Exon 10 expressions from rat, mouse, human and monkey brain cDNA libraries with the common forward primer F2 against Exon 9 and reverse primer R4.2 against Exon 10 (Table S4). (C) GABA-potentiated current rundowns of GABAA receptors containing α1, γ2 and β2L-T365A. (D) The measured currents upon successive additions of GABA expressed as % of the peak current elicited by the first addition of GABA. Significantly different responses between the α1β2L-T365Aγ2L (▾) and α1β2Lγ2L (□) receptors are marked by single (P<0.05) or double (P<0.01) asterisks. (E) Same as part D with 4 mM ATP infusion.
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pone-0006977-g001: Characterizations of Exon 10 of GABRB2.(A) The exonic structure of human GABRB2 showing positions of the tagging SNPs, and the alignment of its Exon 10 with corresponding sequences in other vertebrates (from UCSC Genome Browser). The lengths of the sequences corresponding to the intervening sequences between E9 and E11 in the different species are indicated on the right. Dots represent identities; single line, deletions; double-dashed line,unalignable bases. Exons are shown as numbered boxes. (B) RT-PCR analyses of Exon 10 expressions from rat, mouse, human and monkey brain cDNA libraries with the common forward primer F2 against Exon 9 and reverse primer R4.2 against Exon 10 (Table S4). (C) GABA-potentiated current rundowns of GABAA receptors containing α1, γ2 and β2L-T365A. (D) The measured currents upon successive additions of GABA expressed as % of the peak current elicited by the first addition of GABA. Significantly different responses between the α1β2L-T365Aγ2L (▾) and α1β2Lγ2L (□) receptors are marked by single (P<0.05) or double (P<0.01) asterisks. (E) Same as part D with 4 mM ATP infusion.

Mentions: γ-Aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the vertebrate nervous system. The fast synaptic inhibition is mediated by the opening of a chloride channel formed by the GABAA receptors. The GABAA receptors are also clinically relevant drug targets for anti-convulsant, anxiolytic and sedative-hypnotic agents. Subtypes of GABAA receptors are assembled from pentameric combinations of α1-α6, β1-β3, γ1-γ3, ρ1-ρ3, ε, δ and π subunits [1], [2]. The molecular heterogeneity of GABAA receptor subunits is further increased by the alternative splicing of some subunit mRNAs, producing at least two isoforms of each of the α6, β2, β3 and γ2 subunits [3]. Most GABAA receptors are composed of two α subunits, two β2 subunits and one γ subunit [4]. The β2 subunit gene products of human GABRB2 expressed from cDNA library are found in two alternatively spliced isoforms, the short form β2S and the long form β2L. The inclusion of an extra 38-amino acid Exon 10 in β2L but not in β2S in an intracellular loop brings with it a potential phosphorylation site at Thr365 for calmodulin-dependent protein kinase II (Figure 1A)[5].


Alternative-splicing in the exon-10 region of GABA(A) receptor beta(2) subunit gene: relationships between novel isoforms and psychotic disorders.

Zhao C, Xu Z, Wang F, Chen J, Ng SK, Wong PW, Yu Z, Pun FW, Ren L, Lo WS, Tsang SY, Xue H - PLoS ONE (2009)

Characterizations of Exon 10 of GABRB2.(A) The exonic structure of human GABRB2 showing positions of the tagging SNPs, and the alignment of its Exon 10 with corresponding sequences in other vertebrates (from UCSC Genome Browser). The lengths of the sequences corresponding to the intervening sequences between E9 and E11 in the different species are indicated on the right. Dots represent identities; single line, deletions; double-dashed line,unalignable bases. Exons are shown as numbered boxes. (B) RT-PCR analyses of Exon 10 expressions from rat, mouse, human and monkey brain cDNA libraries with the common forward primer F2 against Exon 9 and reverse primer R4.2 against Exon 10 (Table S4). (C) GABA-potentiated current rundowns of GABAA receptors containing α1, γ2 and β2L-T365A. (D) The measured currents upon successive additions of GABA expressed as % of the peak current elicited by the first addition of GABA. Significantly different responses between the α1β2L-T365Aγ2L (▾) and α1β2Lγ2L (□) receptors are marked by single (P<0.05) or double (P<0.01) asterisks. (E) Same as part D with 4 mM ATP infusion.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2741204&req=5

pone-0006977-g001: Characterizations of Exon 10 of GABRB2.(A) The exonic structure of human GABRB2 showing positions of the tagging SNPs, and the alignment of its Exon 10 with corresponding sequences in other vertebrates (from UCSC Genome Browser). The lengths of the sequences corresponding to the intervening sequences between E9 and E11 in the different species are indicated on the right. Dots represent identities; single line, deletions; double-dashed line,unalignable bases. Exons are shown as numbered boxes. (B) RT-PCR analyses of Exon 10 expressions from rat, mouse, human and monkey brain cDNA libraries with the common forward primer F2 against Exon 9 and reverse primer R4.2 against Exon 10 (Table S4). (C) GABA-potentiated current rundowns of GABAA receptors containing α1, γ2 and β2L-T365A. (D) The measured currents upon successive additions of GABA expressed as % of the peak current elicited by the first addition of GABA. Significantly different responses between the α1β2L-T365Aγ2L (▾) and α1β2Lγ2L (□) receptors are marked by single (P<0.05) or double (P<0.01) asterisks. (E) Same as part D with 4 mM ATP infusion.
Mentions: γ-Aminobutyric acid (GABA) is the major inhibitory amino acid neurotransmitter in the vertebrate nervous system. The fast synaptic inhibition is mediated by the opening of a chloride channel formed by the GABAA receptors. The GABAA receptors are also clinically relevant drug targets for anti-convulsant, anxiolytic and sedative-hypnotic agents. Subtypes of GABAA receptors are assembled from pentameric combinations of α1-α6, β1-β3, γ1-γ3, ρ1-ρ3, ε, δ and π subunits [1], [2]. The molecular heterogeneity of GABAA receptor subunits is further increased by the alternative splicing of some subunit mRNAs, producing at least two isoforms of each of the α6, β2, β3 and γ2 subunits [3]. Most GABAA receptors are composed of two α subunits, two β2 subunits and one γ subunit [4]. The β2 subunit gene products of human GABRB2 expressed from cDNA library are found in two alternatively spliced isoforms, the short form β2S and the long form β2L. The inclusion of an extra 38-amino acid Exon 10 in β2L but not in β2S in an intracellular loop brings with it a potential phosphorylation site at Thr365 for calmodulin-dependent protein kinase II (Figure 1A)[5].

Bottom Line: Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression.Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Applied Genomics Center, Fok Ying Tung Graduate School, The Hong Kong University of Science & Technology, Clear Water Bay, Hong Kong, China.

ABSTRACT

Background: Non-coding single nucleotide polymorphisms (SNPs) in GABRB2, the gene for beta(2)-subunit of gamma-aminobutyric acid type A (GABA(A)) receptor, have been associated with schizophrenia (SCZ) and quantitatively correlated to mRNA expression and alternative splicing.

Methods and findings: Expression of the Exon 10 region of GABRB2 from minigene constructs revealed this region to be an "alternative splicing hotspot" that readily gave rise to differently spliced isoforms depending on intron sequences. This led to a search in human brain cDNA libraries, and the discovery of two novel isoforms, beta(2S1) and beta(2S2), bearing variations in the neighborhood of Exon-10. Quantitative real-time PCR analysis of postmortem brain samples showed increased beta(2S1) expression and decreased beta(2S2) expression in both SCZ and bipolar disorder (BPD) compared to controls. Disease-control differences were significantly correlated with SNP rs187269 in BPD males for both beta(2S1) and beta(2S2) expressions, and significantly correlated with SNPs rs2546620 and rs187269 in SCZ males for beta(2S2) expression. Moreover, site-directed mutagenesis indicated that Thr(365), a potential phosphorylation site in Exon-10, played a key role in determining the time profile of the ATP-dependent electrophysiological current run-down.

Conclusion: This study therefore provided experimental evidence for the importance of non-coding sequences in the Exon-10 region in GABRB2 with respect to beta(2)-subunit splicing diversity and the etiologies of SCZ and BPD.

Show MeSH
Related in: MedlinePlus