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RNA gain-of-function in spinocerebellar ataxia type 8.

Daughters RS, Tuttle DL, Gao W, Ikeda Y, Moseley ML, Ebner TJ, Swanson MS, Ranum LP - PLoS Genet. (2009)

Bottom Line: Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins.These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders.Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUG(exp)) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAG(exp) transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUG(exp) transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUG(exp) transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders. Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

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Endogenous GABT4 up-regulation in SK-N-SH cells induced by CUGexp but not CAGexp transcripts.(A) Schematic of constructs used to express SCA8 CUGexp or CAGexp and pure CUGexp and CAGexp transcripts. (B) qRT-PCR shows increased GABT4 RNA levels in SK-N-SH cells expressing Exon A CUGexp or pure polyCUGexp transcripts. * = statistical significance, p<0.0001. (C) Alternative splicing shifts favoring exon 7 inclusion in cells expressing SCA8 Exon A CUGexp or pure polyCUGexp that does not occur in response to SCA8 Exon A CAG or pure polyCAGexp transcripts compared to vector alone.
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pgen-1000600-g006: Endogenous GABT4 up-regulation in SK-N-SH cells induced by CUGexp but not CAGexp transcripts.(A) Schematic of constructs used to express SCA8 CUGexp or CAGexp and pure CUGexp and CAGexp transcripts. (B) qRT-PCR shows increased GABT4 RNA levels in SK-N-SH cells expressing Exon A CUGexp or pure polyCUGexp transcripts. * = statistical significance, p<0.0001. (C) Alternative splicing shifts favoring exon 7 inclusion in cells expressing SCA8 Exon A CUGexp or pure polyCUGexp that does not occur in response to SCA8 Exon A CAG or pure polyCAGexp transcripts compared to vector alone.

Mentions: To test directly if increases in GABT4 are induced by SCA8 CUGexp or CAGexp transcripts, we examined their effects in human neuroblastoma SK-N-SH cells. Transient transfections were performed using minigenes (Figure 6A) designed to express SCA8 CUG (SCA8-CTGexp) or CAG expansion transcripts (SCA8-REV CAGexp). Cells expressing SCA8 CUGexp transcripts show significant increases in GABT4 RNA levels by qRT-PCR relative to untransfected cells (p = 0.007) or to cells transfected with vector alone (p = 0.006) while expression of the SCA8 CAGexp construct compared to vector alone had no effect (p = 0.72) (Figure 6B). Additionally, transient transfections of minigenes without ATXN8 and ATXN8OS flanking sequence show poly-CUG105 but not poly-CAG105 transcripts up-regulate GABT4 RNA (p = 0.001) (Figure 6C). Further analysis using primers flanking exon 7, show cells expressing higher levels GABT4 transcripts also preferentially express higher ratios of exon 7 included transcripts. (Figure 6B and 6C).


RNA gain-of-function in spinocerebellar ataxia type 8.

Daughters RS, Tuttle DL, Gao W, Ikeda Y, Moseley ML, Ebner TJ, Swanson MS, Ranum LP - PLoS Genet. (2009)

Endogenous GABT4 up-regulation in SK-N-SH cells induced by CUGexp but not CAGexp transcripts.(A) Schematic of constructs used to express SCA8 CUGexp or CAGexp and pure CUGexp and CAGexp transcripts. (B) qRT-PCR shows increased GABT4 RNA levels in SK-N-SH cells expressing Exon A CUGexp or pure polyCUGexp transcripts. * = statistical significance, p<0.0001. (C) Alternative splicing shifts favoring exon 7 inclusion in cells expressing SCA8 Exon A CUGexp or pure polyCUGexp that does not occur in response to SCA8 Exon A CAG or pure polyCAGexp transcripts compared to vector alone.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719092&req=5

pgen-1000600-g006: Endogenous GABT4 up-regulation in SK-N-SH cells induced by CUGexp but not CAGexp transcripts.(A) Schematic of constructs used to express SCA8 CUGexp or CAGexp and pure CUGexp and CAGexp transcripts. (B) qRT-PCR shows increased GABT4 RNA levels in SK-N-SH cells expressing Exon A CUGexp or pure polyCUGexp transcripts. * = statistical significance, p<0.0001. (C) Alternative splicing shifts favoring exon 7 inclusion in cells expressing SCA8 Exon A CUGexp or pure polyCUGexp that does not occur in response to SCA8 Exon A CAG or pure polyCAGexp transcripts compared to vector alone.
Mentions: To test directly if increases in GABT4 are induced by SCA8 CUGexp or CAGexp transcripts, we examined their effects in human neuroblastoma SK-N-SH cells. Transient transfections were performed using minigenes (Figure 6A) designed to express SCA8 CUG (SCA8-CTGexp) or CAG expansion transcripts (SCA8-REV CAGexp). Cells expressing SCA8 CUGexp transcripts show significant increases in GABT4 RNA levels by qRT-PCR relative to untransfected cells (p = 0.007) or to cells transfected with vector alone (p = 0.006) while expression of the SCA8 CAGexp construct compared to vector alone had no effect (p = 0.72) (Figure 6B). Additionally, transient transfections of minigenes without ATXN8 and ATXN8OS flanking sequence show poly-CUG105 but not poly-CAG105 transcripts up-regulate GABT4 RNA (p = 0.001) (Figure 6C). Further analysis using primers flanking exon 7, show cells expressing higher levels GABT4 transcripts also preferentially express higher ratios of exon 7 included transcripts. (Figure 6B and 6C).

Bottom Line: Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins.These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders.Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA.

ABSTRACT
Microsatellite expansions cause a number of dominantly-inherited neurological diseases. Expansions in coding-regions cause protein gain-of-function effects, while non-coding expansions produce toxic RNAs that alter RNA splicing activities of MBNL and CELF proteins. Bi-directional expression of the spinocerebellar ataxia type 8 (SCA8) CTG CAG expansion produces CUG expansion RNAs (CUG(exp)) from the ATXN8OS gene and a nearly pure polyglutamine expansion protein encoded by ATXN8 CAG(exp) transcripts expressed in the opposite direction. Here, we present three lines of evidence that RNA gain-of-function plays a significant role in SCA8: 1) CUG(exp) transcripts accumulate as ribonuclear inclusions that co-localize with MBNL1 in selected neurons in the brain; 2) loss of Mbnl1 enhances motor deficits in SCA8 mice; 3) SCA8 CUG(exp) transcripts trigger splicing changes and increased expression of the CUGBP1-MBNL1 regulated CNS target, GABA-A transporter 4 (GAT4/Gabt4). In vivo optical imaging studies in SCA8 mice confirm that Gabt4 upregulation is associated with the predicted loss of GABAergic inhibition within the granular cell layer. These data demonstrate that CUG(exp) transcripts dysregulate MBNL/CELF regulated pathways in the brain and provide mechanistic insight into the CNS effects of other CUG(exp) disorders. Moreover, our demonstration that relatively short CUG(exp) transcripts cause RNA gain-of-function effects and the growing number of antisense transcripts recently reported in mammalian genomes suggest unrecognized toxic RNAs contribute to the pathophysiology of polyglutamine CAG CTG disorders.

Show MeSH
Related in: MedlinePlus