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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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The NMD pathway modulates expanded CUG transcripts degradation and nuclear foci accumulation. (A) Fluorescent microscopy images of 2d old adult animals expressing either 123CUG repeats or 0CUG in the backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Scale bars correspond to 200μm. (B) qRT-PCR assay for gfp levels in animals expressing either 123CUG repeats or the control GFP in different backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Wild type=1.0. Error bars represent SEM for three biological replicates. (C) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. The strains imaged are 123CUG and 0CUG animals, in wild type (wt) and smg-2(qd101). Yellow arrows indicate expanded CUG nuclear foci. (D) Computational analysis of SM-FISH images of 0CUG (green dots), 0CUG in smg mutant backgrounds (blue dots), 123CUG (red dots) and 123CUG in smg mutant backgrounds (black dots).
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Figure 4: The NMD pathway modulates expanded CUG transcripts degradation and nuclear foci accumulation. (A) Fluorescent microscopy images of 2d old adult animals expressing either 123CUG repeats or 0CUG in the backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Scale bars correspond to 200μm. (B) qRT-PCR assay for gfp levels in animals expressing either 123CUG repeats or the control GFP in different backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Wild type=1.0. Error bars represent SEM for three biological replicates. (C) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. The strains imaged are 123CUG and 0CUG animals, in wild type (wt) and smg-2(qd101). Yellow arrows indicate expanded CUG nuclear foci. (D) Computational analysis of SM-FISH images of 0CUG (green dots), 0CUG in smg mutant backgrounds (blue dots), 123CUG (red dots) and 123CUG in smg mutant backgrounds (black dots).

Mentions: smg-2 encodes an RNA helicase and is a conserved component of the nonsense-mediated mRNA decay (NMD) pathway. The NMD pathway is an evolutionary conserved surveillance mechanism that detects mRNAs containing premature stop codons, preventing toxic expression of truncated proteins25. The identification of smg-2 as a modulator of expanded CUG toxicity suggested that the NMD pathway may recognize and target for degradation RNA transcripts with expanded CUG repeats, even in the 3′ UTRs of non-truncated open reading frames. We analyzed the effects of mutations in NMD components on GFP transcripts bearing 123CUG repeats or control 0CUG in muscle cells using smg-1(r861), smg-2(qd101) and smg-6(r896) mutants. We observed that 123CUG animals in the background of any of the smg mutants showed a strong increase in GFP fluorescence signal relative to the parental strain (Fig. 4A). No such change in fluorescence was observed for the control 0CUG animals (Fig. 4A). Quantitative RT-PCR showed that, mRNA levels of gfp bearing 123CUG repeats were increased by several fold: ≈5.3 fold in smg-1(r861), ≈7.8 fold in smg-2(qd101) and ≈10.1 fold in smg-6(r896) backgrounds, compared to wild type (Fig. 4B). However, no significant change was observed in the levels of gfp mRNA without any CUG repeats in the 3′UTR in the different smg mutant backgrounds compared to the wild type (Fig. 4B). Thus the NMD pathway targets the mRNA transcripts containing the expanded CUG repeats for degradation.


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)

The NMD pathway modulates expanded CUG transcripts degradation and nuclear foci accumulation. (A) Fluorescent microscopy images of 2d old adult animals expressing either 123CUG repeats or 0CUG in the backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Scale bars correspond to 200μm. (B) qRT-PCR assay for gfp levels in animals expressing either 123CUG repeats or the control GFP in different backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Wild type=1.0. Error bars represent SEM for three biological replicates. (C) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. The strains imaged are 123CUG and 0CUG animals, in wild type (wt) and smg-2(qd101). Yellow arrows indicate expanded CUG nuclear foci. (D) Computational analysis of SM-FISH images of 0CUG (green dots), 0CUG in smg mutant backgrounds (blue dots), 123CUG (red dots) and 123CUG in smg mutant backgrounds (black dots).
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Figure 4: The NMD pathway modulates expanded CUG transcripts degradation and nuclear foci accumulation. (A) Fluorescent microscopy images of 2d old adult animals expressing either 123CUG repeats or 0CUG in the backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Scale bars correspond to 200μm. (B) qRT-PCR assay for gfp levels in animals expressing either 123CUG repeats or the control GFP in different backgrounds: wild type (wt), smg-2(qd101), smg-1(r861) and smg-6(r896). Wild type=1.0. Error bars represent SEM for three biological replicates. (C) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. The strains imaged are 123CUG and 0CUG animals, in wild type (wt) and smg-2(qd101). Yellow arrows indicate expanded CUG nuclear foci. (D) Computational analysis of SM-FISH images of 0CUG (green dots), 0CUG in smg mutant backgrounds (blue dots), 123CUG (red dots) and 123CUG in smg mutant backgrounds (black dots).
Mentions: smg-2 encodes an RNA helicase and is a conserved component of the nonsense-mediated mRNA decay (NMD) pathway. The NMD pathway is an evolutionary conserved surveillance mechanism that detects mRNAs containing premature stop codons, preventing toxic expression of truncated proteins25. The identification of smg-2 as a modulator of expanded CUG toxicity suggested that the NMD pathway may recognize and target for degradation RNA transcripts with expanded CUG repeats, even in the 3′ UTRs of non-truncated open reading frames. We analyzed the effects of mutations in NMD components on GFP transcripts bearing 123CUG repeats or control 0CUG in muscle cells using smg-1(r861), smg-2(qd101) and smg-6(r896) mutants. We observed that 123CUG animals in the background of any of the smg mutants showed a strong increase in GFP fluorescence signal relative to the parental strain (Fig. 4A). No such change in fluorescence was observed for the control 0CUG animals (Fig. 4A). Quantitative RT-PCR showed that, mRNA levels of gfp bearing 123CUG repeats were increased by several fold: ≈5.3 fold in smg-1(r861), ≈7.8 fold in smg-2(qd101) and ≈10.1 fold in smg-6(r896) backgrounds, compared to wild type (Fig. 4B). However, no significant change was observed in the levels of gfp mRNA without any CUG repeats in the 3′UTR in the different smg mutant backgrounds compared to the wild type (Fig. 4B). Thus the NMD pathway targets the mRNA transcripts containing the expanded CUG repeats for degradation.

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