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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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Antagonistic Regulation of GABT4 by CUGBP1 and MBNL1 in SK-N-SH cells.(A,B) qRT-PCR showing increased GABT4 transcript levels and alternative splicing changes favoring exon 7 inclusion in cells overexpressing CUGexp transcripts or CUG-BP1 but not in cells overexpressing MBNL1 1/41 or CUGexp and MBNL1 1/41. (C) Protein blot showing GABT4 is increased in cells overexpressing CUGexp transcripts or CUG-BP1, but not in cells overexpressing MBNL1/41 alone or MBNL1/41 and CUGexp transcripts.
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pgen-1000600-g007: Antagonistic Regulation of GABT4 by CUGBP1 and MBNL1 in SK-N-SH cells.(A,B) qRT-PCR showing increased GABT4 transcript levels and alternative splicing changes favoring exon 7 inclusion in cells overexpressing CUGexp transcripts or CUG-BP1 but not in cells overexpressing MBNL1 1/41 or CUGexp and MBNL1 1/41. (C) Protein blot showing GABT4 is increased in cells overexpressing CUGexp transcripts or CUG-BP1, but not in cells overexpressing MBNL1/41 alone or MBNL1/41 and CUGexp transcripts.

Mentions: To test if GABT4 expression is antagonistically regulated by CUGBP1 and MBNL1, we transfected SK-N-SH cells using GFP-tagged human MBNL1/41 and CUGBP1 minigenes capable of inducing alternative splicing changes in cell culture [21],[36]. As above, alternatively-spliced exon 7 transcripts were assayed by RT-PCR with primers located in GABT4 exons 6 and 8 and primers spanning exons 1 and 2 were used to assess endogenous levels of GABT4 by qRT-PCR. Cells overexpressing CUGBP1 (p<0.0001) or SCA8 exon A CTGexp minigenes show increases in GABT4 RNA (p = 0.003) and a concomitant increases in protein and a splicing shift favoring exon 7 inclusion compared to vector alone (Figure 7A–7C). While no change in GABT4 RNA or protein was seen in cells overexpressing MBNL1/41 alone, overexpression of MBNL1/41 and SCA8 CUGexp transcripts reverses the increase in GABT4 RNA (p<0.0001) and protein (Figure 7A and 7C) triggered by SCA8 CUGexp transcripts alone and restores exon 7 alternative splicing ratios to the normal adult pattern (Figure 7B). Sequence analysis of the GABT4 RT-PCR products confirm the upper and lower bands include and exclude exon 7 respectively and that the (-) exon 7 transcripts have a premature stop codon in exon 8. Taken together, these results are consistent with a model in which SCA8 CUGexp transcripts alter the regulation of GABT4 by sequestration of MBNL1 and/or an increase in the expression or activity of CUGBP1.


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)

Antagonistic Regulation of GABT4 by CUGBP1 and MBNL1 in SK-N-SH cells.(A,B) qRT-PCR showing increased GABT4 transcript levels and alternative splicing changes favoring exon 7 inclusion in cells overexpressing CUGexp transcripts or CUG-BP1 but not in cells overexpressing MBNL1 1/41 or CUGexp and MBNL1 1/41. (C) Protein blot showing GABT4 is increased in cells overexpressing CUGexp transcripts or CUG-BP1, but not in cells overexpressing MBNL1/41 alone or MBNL1/41 and CUGexp transcripts.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000600-g007: Antagonistic Regulation of GABT4 by CUGBP1 and MBNL1 in SK-N-SH cells.(A,B) qRT-PCR showing increased GABT4 transcript levels and alternative splicing changes favoring exon 7 inclusion in cells overexpressing CUGexp transcripts or CUG-BP1 but not in cells overexpressing MBNL1 1/41 or CUGexp and MBNL1 1/41. (C) Protein blot showing GABT4 is increased in cells overexpressing CUGexp transcripts or CUG-BP1, but not in cells overexpressing MBNL1/41 alone or MBNL1/41 and CUGexp transcripts.
Mentions: To test if GABT4 expression is antagonistically regulated by CUGBP1 and MBNL1, we transfected SK-N-SH cells using GFP-tagged human MBNL1/41 and CUGBP1 minigenes capable of inducing alternative splicing changes in cell culture [21],[36]. As above, alternatively-spliced exon 7 transcripts were assayed by RT-PCR with primers located in GABT4 exons 6 and 8 and primers spanning exons 1 and 2 were used to assess endogenous levels of GABT4 by qRT-PCR. Cells overexpressing CUGBP1 (p<0.0001) or SCA8 exon A CTGexp minigenes show increases in GABT4 RNA (p = 0.003) and a concomitant increases in protein and a splicing shift favoring exon 7 inclusion compared to vector alone (Figure 7A–7C). While no change in GABT4 RNA or protein was seen in cells overexpressing MBNL1/41 alone, overexpression of MBNL1/41 and SCA8 CUGexp transcripts reverses the increase in GABT4 RNA (p<0.0001) and protein (Figure 7A and 7C) triggered by SCA8 CUGexp transcripts alone and restores exon 7 alternative splicing ratios to the normal adult pattern (Figure 7B). Sequence analysis of the GABT4 RT-PCR products confirm the upper and lower bands include and exclude exon 7 respectively and that the (-) exon 7 transcripts have a premature stop codon in exon 8. Taken together, these results are consistent with a model in which SCA8 CUGexp transcripts alter the regulation of GABT4 by sequestration of MBNL1 and/or an increase in the expression or activity of CUGBP1.

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