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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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NMD downregulation causes an increase in CUG repeat mRNA foci number in myotonic dystrophy 1 patient fibroblast cells. (A) SM-FISH of DM1-affected or normal human fibroblast cells in which UPF1 was downregulated relative to control non-transfected or transfected with scrambled siRNAs (mock) cells. The DM1 human fibroblast cell line used expressed the gene dmpk bearing 2000CUG in its 3′UTR. (B) Histogram represents the distribution of the number of foci in DM1 cells that were downregulated for UPF1, mock and non-transfected controls. UPF1 downregulation led to a significant increase in the number of nuclear foci present relative to mock (p<0.0001) and non-transfected cells (p<0.00003), using t-student test. N indicates the total number of cells analyzed. Two independent experiments were performed. Bar, 5μm.
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Figure 6: NMD downregulation causes an increase in CUG repeat mRNA foci number in myotonic dystrophy 1 patient fibroblast cells. (A) SM-FISH of DM1-affected or normal human fibroblast cells in which UPF1 was downregulated relative to control non-transfected or transfected with scrambled siRNAs (mock) cells. The DM1 human fibroblast cell line used expressed the gene dmpk bearing 2000CUG in its 3′UTR. (B) Histogram represents the distribution of the number of foci in DM1 cells that were downregulated for UPF1, mock and non-transfected controls. UPF1 downregulation led to a significant increase in the number of nuclear foci present relative to mock (p<0.0001) and non-transfected cells (p<0.00003), using t-student test. N indicates the total number of cells analyzed. Two independent experiments were performed. Bar, 5μm.

Mentions: To establish whether NMD recognition of expanded CUG repeats is a conserved cellular mechanism, we analyzed the nuclear RNA foci phenotype of NMD gene inactivations in human DM1 patient fibroblast cells expressing 2000 CUG repeats in the DMPK1 mRNA, as well as in control fibroblasts expressing a DMPK1 mRNA with 7 to 35 such CUG repeats. We tested for changes in foci number when the human orthologue of smg-2, UPF1, was inactivated by RNAi. We used SM-FISH for RNA foci detection, with 5 probes complementary to the CUG repeat region and 23 probes complementary to the last three exons of DMPK1 which are not composed of CUG repeats. UPF1 was down-regulated using siRNAs in DM1 and in normal fibroblasts and these cells were analyzed by SM-FISH 24h post siRNA-transfection. For both control fibroblasts and fibroblasts isolated from DM1 patients, UPF1 siRNAs decreased UPF1 protein levels by 35%–40% compared to scrambled siRNAs (Supplementary Fig. 7C, D). There was lower cell recovery after UPF1 knockdown, suggesting that knockdown of NMD components may cause a loss of cell viability, deflating the measured level of UPF1 knockdown. But even with the modest UPF1 knockdown, SM-FISH analysis revealed an increase in the number of nuclear foci in DM1 cells treated with UPF1 siRNAs compared to untreated DM1 cells or DM1 cells treated with mock siRNAs (Fig. 6A). In contrast, normal fibroblast cells bearing just a few CUG repeats in the DMPK gene exhibited no nuclear foci in both untreated or treated with UPF1 siRNAs (Fig. 6A). The number of foci present in the DM1 cells was quantified (see Online Methods) and UPF1 down-regulation caused a significant increase in the percentage of cells containing a higher number of foci (Fig. 6B). Our data supports a conserved role for NMD in the identification of transcripts bearing GC-rich sequences in their 3′UTR. Furthermore, our results support the function of NMD as an important element in the toxicity of expanded CUG repeat transcripts in myotonic dystrophy 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)

NMD downregulation causes an increase in CUG repeat mRNA foci number in myotonic dystrophy 1 patient fibroblast cells. (A) SM-FISH of DM1-affected or normal human fibroblast cells in which UPF1 was downregulated relative to control non-transfected or transfected with scrambled siRNAs (mock) cells. The DM1 human fibroblast cell line used expressed the gene dmpk bearing 2000CUG in its 3′UTR. (B) Histogram represents the distribution of the number of foci in DM1 cells that were downregulated for UPF1, mock and non-transfected controls. UPF1 downregulation led to a significant increase in the number of nuclear foci present relative to mock (p<0.0001) and non-transfected cells (p<0.00003), using t-student test. N indicates the total number of cells analyzed. Two independent experiments were performed. Bar, 5μm.
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Figure 6: NMD downregulation causes an increase in CUG repeat mRNA foci number in myotonic dystrophy 1 patient fibroblast cells. (A) SM-FISH of DM1-affected or normal human fibroblast cells in which UPF1 was downregulated relative to control non-transfected or transfected with scrambled siRNAs (mock) cells. The DM1 human fibroblast cell line used expressed the gene dmpk bearing 2000CUG in its 3′UTR. (B) Histogram represents the distribution of the number of foci in DM1 cells that were downregulated for UPF1, mock and non-transfected controls. UPF1 downregulation led to a significant increase in the number of nuclear foci present relative to mock (p<0.0001) and non-transfected cells (p<0.00003), using t-student test. N indicates the total number of cells analyzed. Two independent experiments were performed. Bar, 5μm.
Mentions: To establish whether NMD recognition of expanded CUG repeats is a conserved cellular mechanism, we analyzed the nuclear RNA foci phenotype of NMD gene inactivations in human DM1 patient fibroblast cells expressing 2000 CUG repeats in the DMPK1 mRNA, as well as in control fibroblasts expressing a DMPK1 mRNA with 7 to 35 such CUG repeats. We tested for changes in foci number when the human orthologue of smg-2, UPF1, was inactivated by RNAi. We used SM-FISH for RNA foci detection, with 5 probes complementary to the CUG repeat region and 23 probes complementary to the last three exons of DMPK1 which are not composed of CUG repeats. UPF1 was down-regulated using siRNAs in DM1 and in normal fibroblasts and these cells were analyzed by SM-FISH 24h post siRNA-transfection. For both control fibroblasts and fibroblasts isolated from DM1 patients, UPF1 siRNAs decreased UPF1 protein levels by 35%–40% compared to scrambled siRNAs (Supplementary Fig. 7C, D). There was lower cell recovery after UPF1 knockdown, suggesting that knockdown of NMD components may cause a loss of cell viability, deflating the measured level of UPF1 knockdown. But even with the modest UPF1 knockdown, SM-FISH analysis revealed an increase in the number of nuclear foci in DM1 cells treated with UPF1 siRNAs compared to untreated DM1 cells or DM1 cells treated with mock siRNAs (Fig. 6A). In contrast, normal fibroblast cells bearing just a few CUG repeats in the DMPK gene exhibited no nuclear foci in both untreated or treated with UPF1 siRNAs (Fig. 6A). The number of foci present in the DM1 cells was quantified (see Online Methods) and UPF1 down-regulation caused a significant increase in the percentage of cells containing a higher number of foci (Fig. 6B). Our data supports a conserved role for NMD in the identification of transcripts bearing GC-rich sequences in their 3′UTR. Furthermore, our results support the function of NMD as an important element in the toxicity of expanded CUG repeat transcripts in myotonic dystrophy 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