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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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3′UTR CUG repeat sequence composition triggers NMD recognition for degradation. Fluorescent microscopy images of the strains 123CUG, GC-rich and AT-rich, in different RNAi gene inactivations: empty vector control (ctrl), smg-1, smg-2, and smg-6. Images of 3d old adult animals. Bar, 200μm.
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Figure 5: 3′UTR CUG repeat sequence composition triggers NMD recognition for degradation. Fluorescent microscopy images of the strains 123CUG, GC-rich and AT-rich, in different RNAi gene inactivations: empty vector control (ctrl), smg-1, smg-2, and smg-6. Images of 3d old adult animals. Bar, 200μm.

Mentions: To examine whether the skewed sequence composition of expanded CUG repeat sequences targets them for the NMD pathway, we explored the influence of GC composition on NMD. 3′UTRs are typically A/U rich (≈65–70% AT-rich), exhibiting a nucleotide composition distinct from coding (≈50–55% AT-rich) or intergenic regions26,27. The let-858 3′ UTR to which the 123 CUG repeat was added is 384 nucleotides and 30% GC. The added CUG repeat elements are rich in G and C nucleotides (≈66%) that may contribute to the recognition by the NMD pathway. We generated expression plasmids in which the 3′UTR (CTG)n sequence was substituted by a non-repeat sequence with either a 66% or 34% GC nucleotide content (Supplementary Fig. 7A, B). The DNA sequences used were cloned from non-C. elegans organisms or from entirely synthetic nucleotide sequences bearing similar GC percentages to avoid a possible recognition of endogenous signal sequences (Supplementary Notes). GFP reporter genes bearing GC-rich 3′ UTR elements from non-C. elegans organisms exhibited weaker GFP fluorescence, or no fluorescence at all in the case of synthetic sequences, compared to those bearing the corresponding AT-rich elements (Fig. 5, Supplementary Fig. 7B). Strains expressing GC-rich elements from a non-C. elegans genome placed in the 3′UTR of the GFP reporter gene showed a significant increase in fluorescence when either smg-1 or smg-2 were inactivated by RNAi, whereas no change in GFP intensity was detected for AT-rich (Fig. 5, Supplementary Fig. 7A, B). Fusion genes engineered with synthetic, random high GC percentage sequences showed a stronger increase in fluorescence in the smg-2 background relative to two regulators of smg-2 phosphorylation smg-1 or smg-6 (Supplementary Fig. 7A, B). These data demonstrate that the results observed for the GC-rich versus AT-rich sequences were not due to a sequence-specific endogenous 3′ UTR identity signal present in the sequence used. These results further establish that the increase in distance between the stop codon and the polyA signal due to the addition of the CUG repeat sequence does not contribute to NMD recognition, since no repression was observed for AT-rich transcripts. These data support a model in which mRNAs, containing CUG repeats in their 3′UTR, are NMD substrates. Furthermore, it reveals that the NMD recognition of CUG-containing mRNA is dependent on nucleotide composition, either due to the presence of a GC-rich sequence in a region usually A/U-rich, or due to the formation of specific secondary structures associated to the presence of these nucleotides. While both the GC-rich 3′ UTR element and the 123CUG repeat element reporter genes are responsive to disruption of the NMD pathway, none of the 15 gene inactivations that strongly disable 123CUG repeat repression in muscle disrupt the repression conferred by GC-rich element. Thus, the detection and localization to foci of 123CUG repeats by these genes is distinct from the detection and degradation of GC rich elements by the NMD system.


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)

3′UTR CUG repeat sequence composition triggers NMD recognition for degradation. Fluorescent microscopy images of the strains 123CUG, GC-rich and AT-rich, in different RNAi gene inactivations: empty vector control (ctrl), smg-1, smg-2, and smg-6. Images of 3d old adult animals. Bar, 200μm.
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Figure 5: 3′UTR CUG repeat sequence composition triggers NMD recognition for degradation. Fluorescent microscopy images of the strains 123CUG, GC-rich and AT-rich, in different RNAi gene inactivations: empty vector control (ctrl), smg-1, smg-2, and smg-6. Images of 3d old adult animals. Bar, 200μm.
Mentions: To examine whether the skewed sequence composition of expanded CUG repeat sequences targets them for the NMD pathway, we explored the influence of GC composition on NMD. 3′UTRs are typically A/U rich (≈65–70% AT-rich), exhibiting a nucleotide composition distinct from coding (≈50–55% AT-rich) or intergenic regions26,27. The let-858 3′ UTR to which the 123 CUG repeat was added is 384 nucleotides and 30% GC. The added CUG repeat elements are rich in G and C nucleotides (≈66%) that may contribute to the recognition by the NMD pathway. We generated expression plasmids in which the 3′UTR (CTG)n sequence was substituted by a non-repeat sequence with either a 66% or 34% GC nucleotide content (Supplementary Fig. 7A, B). The DNA sequences used were cloned from non-C. elegans organisms or from entirely synthetic nucleotide sequences bearing similar GC percentages to avoid a possible recognition of endogenous signal sequences (Supplementary Notes). GFP reporter genes bearing GC-rich 3′ UTR elements from non-C. elegans organisms exhibited weaker GFP fluorescence, or no fluorescence at all in the case of synthetic sequences, compared to those bearing the corresponding AT-rich elements (Fig. 5, Supplementary Fig. 7B). Strains expressing GC-rich elements from a non-C. elegans genome placed in the 3′UTR of the GFP reporter gene showed a significant increase in fluorescence when either smg-1 or smg-2 were inactivated by RNAi, whereas no change in GFP intensity was detected for AT-rich (Fig. 5, Supplementary Fig. 7A, B). Fusion genes engineered with synthetic, random high GC percentage sequences showed a stronger increase in fluorescence in the smg-2 background relative to two regulators of smg-2 phosphorylation smg-1 or smg-6 (Supplementary Fig. 7A, B). These data demonstrate that the results observed for the GC-rich versus AT-rich sequences were not due to a sequence-specific endogenous 3′ UTR identity signal present in the sequence used. These results further establish that the increase in distance between the stop codon and the polyA signal due to the addition of the CUG repeat sequence does not contribute to NMD recognition, since no repression was observed for AT-rich transcripts. These data support a model in which mRNAs, containing CUG repeats in their 3′UTR, are NMD substrates. Furthermore, it reveals that the NMD recognition of CUG-containing mRNA is dependent on nucleotide composition, either due to the presence of a GC-rich sequence in a region usually A/U-rich, or due to the formation of specific secondary structures associated to the presence of these nucleotides. While both the GC-rich 3′ UTR element and the 123CUG repeat element reporter genes are responsive to disruption of the NMD pathway, none of the 15 gene inactivations that strongly disable 123CUG repeat repression in muscle disrupt the repression conferred by GC-rich element. Thus, the detection and localization to foci of 123CUG repeats by these genes is distinct from the detection and degradation of GC rich elements by the NMD system.

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