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Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans.

Garcia SM, Tabach Y, Lourenço GF, Armakola M, Ruvkun G - Nat. Struct. Mol. Biol. (2014)

Bottom Line: Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions.A subset of the genes are also involved in other degenerative disorders.Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions. Here we used Caenorhabditis elegans expressing CUG repeats to identify genes that modulate the toxicity of such repeats. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and that modulate formation of nuclear foci by CUG-repeat RNA. These genes regulate CUG repeat-induced toxicity through distinct mechanisms including RNA export and clearance, thus suggesting that CUG-repeat toxicity is mediated by multiple pathways. A subset of the genes are also involved in other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway has a conserved role in regulating CUG-repeat-RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3'-untranslated-region GC-nucleotide content. Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

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Expanded CUG-dependent C. elegans muscle phenotypes(A) Diagram of CUG-containing plasmids for expression in C. elegans muscle cells, under the myo-3 promoter. n indicates number of CUG repeats. (B) Quantification of GFP expression levels from reporter genes with 123CUG repeats or 0 CUG repeats in the 3′UTR, relative to actin. Graph shows mean and s.d. for 3 independent experiments, p was determined by Student’s t test. Bottom, western blots using GFP and actin antibodies, actin was used for sample normalization. (C) Motility assays for 6d adults. Data plotted corresponds to average percentage of population to reach food at each time point. Error bars represent SD from at least 3 independent experiments; in each experiment, 3–5 replicas of ca. 100–150 animals were analyzed. (D) Confocal single molecule RNA fluorescence in situ hybridization (SM-FISH) images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI (blue). Yellow arrows indicate expanded CUG nuclear foci, and the red asterisk (●) indicates the nucleolus. (E) Computational analysis of SM-FISH muscle cell images of 0CUG, 8CUG and 123CUG animals. Each dot corresponds to an analyzed SM-FISH image. The red dotted square indicates the region of clustering of the 123CUG images (red dots). (F) confocal SM-FISH images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI and mCherry fluorescence is shown on the right. The strains express GFP with 123CUG or 0CUG in a mCHERRY or MBL-1::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci. MBL-1::mCHERRY localizes to the nucleus. (G) Computational analysis of SM-FISH images of 0CUG, 0CUG;mbl-1::mCherry, 123CUG and 123CUG;mbl-1::mCherry animals.
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Figure 1: Expanded CUG-dependent C. elegans muscle phenotypes(A) Diagram of CUG-containing plasmids for expression in C. elegans muscle cells, under the myo-3 promoter. n indicates number of CUG repeats. (B) Quantification of GFP expression levels from reporter genes with 123CUG repeats or 0 CUG repeats in the 3′UTR, relative to actin. Graph shows mean and s.d. for 3 independent experiments, p was determined by Student’s t test. Bottom, western blots using GFP and actin antibodies, actin was used for sample normalization. (C) Motility assays for 6d adults. Data plotted corresponds to average percentage of population to reach food at each time point. Error bars represent SD from at least 3 independent experiments; in each experiment, 3–5 replicas of ca. 100–150 animals were analyzed. (D) Confocal single molecule RNA fluorescence in situ hybridization (SM-FISH) images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI (blue). Yellow arrows indicate expanded CUG nuclear foci, and the red asterisk (●) indicates the nucleolus. (E) Computational analysis of SM-FISH muscle cell images of 0CUG, 8CUG and 123CUG animals. Each dot corresponds to an analyzed SM-FISH image. The red dotted square indicates the region of clustering of the 123CUG images (red dots). (F) confocal SM-FISH images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI and mCherry fluorescence is shown on the right. The strains express GFP with 123CUG or 0CUG in a mCHERRY or MBL-1::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci. MBL-1::mCHERRY localizes to the nucleus. (G) Computational analysis of SM-FISH images of 0CUG, 0CUG;mbl-1::mCherry, 123CUG and 123CUG;mbl-1::mCherry animals.

Mentions: We generated a set of C. elegans reporter genes expressing GFP with 3′UTR containing various lengths of CTG repeats in body wall muscle cells, using the myo-3 muscle-specific promoter (Fig. 1A). Reporter constructs without any CUG repeats in the 384-nt 3′UTR from the let-858 gene (0CUG) displayed strong GFP fluorescence at all developmental stages, with a modest decline during adulthood. Analogous constructs with eight CUG repeats showed similar results with mild changes in GFP fluorescence (data not shown). In contrast, the presence of 123 CUG repeats in the 3′UTR (123CUG, a pathogenic repeat length in mammalian myocytes) resulted in a sharp decline in GFP fluorescence as animals developed to adults. Western blotting analyses revealed a sharp decrease in GFP protein levels in 3 day (3d) old adult stage animals of the 123CUG strain (12% compared to protein levels at the L2 larval stage). The 3d adult stage animals of control 0CUG strain showed 50% of the GFP levels in L2) (Fig. 1B). We used the decline in adult stage GFP fluorescence in123CUG transgenic animals for RNAi screens to identify genes that influence toxicity of expanded CUG repeats.


Identification of genes in toxicity pathways of trinucleotide-repeat RNA in C. elegans.

Garcia SM, Tabach Y, Lourenço GF, Armakola M, Ruvkun G - Nat. Struct. Mol. Biol. (2014)

Expanded CUG-dependent C. elegans muscle phenotypes(A) Diagram of CUG-containing plasmids for expression in C. elegans muscle cells, under the myo-3 promoter. n indicates number of CUG repeats. (B) Quantification of GFP expression levels from reporter genes with 123CUG repeats or 0 CUG repeats in the 3′UTR, relative to actin. Graph shows mean and s.d. for 3 independent experiments, p was determined by Student’s t test. Bottom, western blots using GFP and actin antibodies, actin was used for sample normalization. (C) Motility assays for 6d adults. Data plotted corresponds to average percentage of population to reach food at each time point. Error bars represent SD from at least 3 independent experiments; in each experiment, 3–5 replicas of ca. 100–150 animals were analyzed. (D) Confocal single molecule RNA fluorescence in situ hybridization (SM-FISH) images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI (blue). Yellow arrows indicate expanded CUG nuclear foci, and the red asterisk (●) indicates the nucleolus. (E) Computational analysis of SM-FISH muscle cell images of 0CUG, 8CUG and 123CUG animals. Each dot corresponds to an analyzed SM-FISH image. The red dotted square indicates the region of clustering of the 123CUG images (red dots). (F) confocal SM-FISH images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI and mCherry fluorescence is shown on the right. The strains express GFP with 123CUG or 0CUG in a mCHERRY or MBL-1::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci. MBL-1::mCHERRY localizes to the nucleus. (G) Computational analysis of SM-FISH images of 0CUG, 0CUG;mbl-1::mCherry, 123CUG and 123CUG;mbl-1::mCherry animals.
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Related In: Results  -  Collection

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Figure 1: Expanded CUG-dependent C. elegans muscle phenotypes(A) Diagram of CUG-containing plasmids for expression in C. elegans muscle cells, under the myo-3 promoter. n indicates number of CUG repeats. (B) Quantification of GFP expression levels from reporter genes with 123CUG repeats or 0 CUG repeats in the 3′UTR, relative to actin. Graph shows mean and s.d. for 3 independent experiments, p was determined by Student’s t test. Bottom, western blots using GFP and actin antibodies, actin was used for sample normalization. (C) Motility assays for 6d adults. Data plotted corresponds to average percentage of population to reach food at each time point. Error bars represent SD from at least 3 independent experiments; in each experiment, 3–5 replicas of ca. 100–150 animals were analyzed. (D) Confocal single molecule RNA fluorescence in situ hybridization (SM-FISH) images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI (blue). Yellow arrows indicate expanded CUG nuclear foci, and the red asterisk (●) indicates the nucleolus. (E) Computational analysis of SM-FISH muscle cell images of 0CUG, 8CUG and 123CUG animals. Each dot corresponds to an analyzed SM-FISH image. The red dotted square indicates the region of clustering of the 123CUG images (red dots). (F) confocal SM-FISH images of C. elegans muscle cells for GFP RNA transcripts (right, white); nucleus are stained with DAPI and mCherry fluorescence is shown on the right. The strains express GFP with 123CUG or 0CUG in a mCHERRY or MBL-1::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci. MBL-1::mCHERRY localizes to the nucleus. (G) Computational analysis of SM-FISH images of 0CUG, 0CUG;mbl-1::mCherry, 123CUG and 123CUG;mbl-1::mCherry animals.
Mentions: We generated a set of C. elegans reporter genes expressing GFP with 3′UTR containing various lengths of CTG repeats in body wall muscle cells, using the myo-3 muscle-specific promoter (Fig. 1A). Reporter constructs without any CUG repeats in the 384-nt 3′UTR from the let-858 gene (0CUG) displayed strong GFP fluorescence at all developmental stages, with a modest decline during adulthood. Analogous constructs with eight CUG repeats showed similar results with mild changes in GFP fluorescence (data not shown). In contrast, the presence of 123 CUG repeats in the 3′UTR (123CUG, a pathogenic repeat length in mammalian myocytes) resulted in a sharp decline in GFP fluorescence as animals developed to adults. Western blotting analyses revealed a sharp decrease in GFP protein levels in 3 day (3d) old adult stage animals of the 123CUG strain (12% compared to protein levels at the L2 larval stage). The 3d adult stage animals of control 0CUG strain showed 50% of the GFP levels in L2) (Fig. 1B). We used the decline in adult stage GFP fluorescence in123CUG transgenic animals for RNAi screens to identify genes that influence toxicity of expanded CUG repeats.

Bottom Line: Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions.A subset of the genes are also involved in other degenerative disorders.Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA. [2] Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

ABSTRACT
Myotonic dystrophy disorders are caused by expanded CUG repeats in noncoding regions. Here we used Caenorhabditis elegans expressing CUG repeats to identify genes that modulate the toxicity of such repeats. We identified 15 conserved genes that function as suppressors or enhancers of CUG repeat-induced toxicity and that modulate formation of nuclear foci by CUG-repeat RNA. These genes regulate CUG repeat-induced toxicity through distinct mechanisms including RNA export and clearance, thus suggesting that CUG-repeat toxicity is mediated by multiple pathways. A subset of the genes are also involved in other degenerative disorders. The nonsense-mediated mRNA decay (NMD) pathway has a conserved role in regulating CUG-repeat-RNA transcript levels and toxicity, and NMD recognition of toxic RNAs depends on 3'-untranslated-region GC-nucleotide content. Our studies suggest a broader surveillance role for NMD in which variations in this pathway influence multiple degenerative diseases.

Show MeSH
Related in: MedlinePlus