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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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Identification of gene inactivation that modulate expanded CUG repeat toxicity (A) Gene inactivations that disrupt the late stage down-regulation of GFP fluorescence mediated by 123 CUG repeats in the 3′ UTR. Fluorescent microscopy images of the strains 123CUG and the control 0CUG, on different RNAi gene inactivations: empty vector control (ctrl), npp-4, hda-1, C06A1.6 and smg-2. Images were taken at the 3d old adult stage. Bar, 200μm. (B) Genetic suppressors and enhancers of expanded CUG repeat toxicity. Graph of velocity measurements of 0CUG (grey) and 123CUG (white) animals fed on different gene inactivations. The plotted velocities (μm/sec) correspond to the median of at least two experiments, where the red bars correspond to strains fed on control vector. Red line indicates the median velocity, and white shading represents the 25th and 75th percentile for the 123CUG animals fed on control vector. The dotted orange line represents the maximum and minimum of the median velocity for 123CUG animals fed on control vector. Indicated by red asterisk (*) are the significant gene inactivations, where significance was determined by Kolmogorov-Smirnov p-value. The black asterisk indicates the gene smg-2.
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Figure 2: Identification of gene inactivation that modulate expanded CUG repeat toxicity (A) Gene inactivations that disrupt the late stage down-regulation of GFP fluorescence mediated by 123 CUG repeats in the 3′ UTR. Fluorescent microscopy images of the strains 123CUG and the control 0CUG, on different RNAi gene inactivations: empty vector control (ctrl), npp-4, hda-1, C06A1.6 and smg-2. Images were taken at the 3d old adult stage. Bar, 200μm. (B) Genetic suppressors and enhancers of expanded CUG repeat toxicity. Graph of velocity measurements of 0CUG (grey) and 123CUG (white) animals fed on different gene inactivations. The plotted velocities (μm/sec) correspond to the median of at least two experiments, where the red bars correspond to strains fed on control vector. Red line indicates the median velocity, and white shading represents the 25th and 75th percentile for the 123CUG animals fed on control vector. The dotted orange line represents the maximum and minimum of the median velocity for 123CUG animals fed on control vector. Indicated by red asterisk (*) are the significant gene inactivations, where significance was determined by Kolmogorov-Smirnov p-value. The black asterisk indicates the gene smg-2.

Mentions: To identify genes that mediate expanded CUG repeat RNA pathogenesis, we used RNAi to reveal gene inactivations that can modify expanded CUG repeat RNA toxicity. A two-step screen was performed, with an initial fluorescent-based RNAi screen, followed by a secondary motility-based screen on hits from the primary screen (Supplementary Fig. 3A). For the fluorescent-based screen, we assayed for gene inactivations that disrupt the late stage down-regulation of GFP fluorescence specific to the 123CUG strain. We screened an RNAi library of 403 clones targeting genes that encode RNA-binding proteins and factors implicated in small RNA pathways20. This type of sub-library was expected to have a high representation of genes involved in expanded CUG repeat toxicity. Of the 403 genes tested, after re-screening in triplicate, 84 gene inactivations were selected that induced an increase in late developmental stage GFP fluorescence specifically in the 123CUG strain without affecting the control 0CUG strain (Figure 2A, Supplementary Fig. 3B, Supplementary Table 1). We tested each of the 84 gene inactivations identified for their ability to modulate the motility defect observed in 123CUG animals (see Online Methods). The 123CUG animals on the control RNAi showed a severe loss in motility, with a median velocity of ≈17μm/sec, compared to the 0CUG strain on the same control RNAi at ≈100μm/sec (Fig. 2B) similar to wild type animals. We identified 14 gene inactivations that significantly (p<0.01 using the two-sample Kolmogorov-Smirnov test) increased or decreased the velocity of 123CUG animals without affecting the control (0CUG) animals (Fig. 2B, Table 1).


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)

Identification of gene inactivation that modulate expanded CUG repeat toxicity (A) Gene inactivations that disrupt the late stage down-regulation of GFP fluorescence mediated by 123 CUG repeats in the 3′ UTR. Fluorescent microscopy images of the strains 123CUG and the control 0CUG, on different RNAi gene inactivations: empty vector control (ctrl), npp-4, hda-1, C06A1.6 and smg-2. Images were taken at the 3d old adult stage. Bar, 200μm. (B) Genetic suppressors and enhancers of expanded CUG repeat toxicity. Graph of velocity measurements of 0CUG (grey) and 123CUG (white) animals fed on different gene inactivations. The plotted velocities (μm/sec) correspond to the median of at least two experiments, where the red bars correspond to strains fed on control vector. Red line indicates the median velocity, and white shading represents the 25th and 75th percentile for the 123CUG animals fed on control vector. The dotted orange line represents the maximum and minimum of the median velocity for 123CUG animals fed on control vector. Indicated by red asterisk (*) are the significant gene inactivations, where significance was determined by Kolmogorov-Smirnov p-value. The black asterisk indicates the gene smg-2.
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Related In: Results  -  Collection

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Figure 2: Identification of gene inactivation that modulate expanded CUG repeat toxicity (A) Gene inactivations that disrupt the late stage down-regulation of GFP fluorescence mediated by 123 CUG repeats in the 3′ UTR. Fluorescent microscopy images of the strains 123CUG and the control 0CUG, on different RNAi gene inactivations: empty vector control (ctrl), npp-4, hda-1, C06A1.6 and smg-2. Images were taken at the 3d old adult stage. Bar, 200μm. (B) Genetic suppressors and enhancers of expanded CUG repeat toxicity. Graph of velocity measurements of 0CUG (grey) and 123CUG (white) animals fed on different gene inactivations. The plotted velocities (μm/sec) correspond to the median of at least two experiments, where the red bars correspond to strains fed on control vector. Red line indicates the median velocity, and white shading represents the 25th and 75th percentile for the 123CUG animals fed on control vector. The dotted orange line represents the maximum and minimum of the median velocity for 123CUG animals fed on control vector. Indicated by red asterisk (*) are the significant gene inactivations, where significance was determined by Kolmogorov-Smirnov p-value. The black asterisk indicates the gene smg-2.
Mentions: To identify genes that mediate expanded CUG repeat RNA pathogenesis, we used RNAi to reveal gene inactivations that can modify expanded CUG repeat RNA toxicity. A two-step screen was performed, with an initial fluorescent-based RNAi screen, followed by a secondary motility-based screen on hits from the primary screen (Supplementary Fig. 3A). For the fluorescent-based screen, we assayed for gene inactivations that disrupt the late stage down-regulation of GFP fluorescence specific to the 123CUG strain. We screened an RNAi library of 403 clones targeting genes that encode RNA-binding proteins and factors implicated in small RNA pathways20. This type of sub-library was expected to have a high representation of genes involved in expanded CUG repeat toxicity. Of the 403 genes tested, after re-screening in triplicate, 84 gene inactivations were selected that induced an increase in late developmental stage GFP fluorescence specifically in the 123CUG strain without affecting the control 0CUG strain (Figure 2A, Supplementary Fig. 3B, Supplementary Table 1). We tested each of the 84 gene inactivations identified for their ability to modulate the motility defect observed in 123CUG animals (see Online Methods). The 123CUG animals on the control RNAi showed a severe loss in motility, with a median velocity of ≈17μm/sec, compared to the 0CUG strain on the same control RNAi at ≈100μm/sec (Fig. 2B) similar to wild type animals. We identified 14 gene inactivations that significantly (p<0.01 using the two-sample Kolmogorov-Smirnov test) increased or decreased the velocity of 123CUG animals without affecting the control (0CUG) animals (Fig. 2B, Table 1).

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