Limits...
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.

Show MeSH

Related in: MedlinePlus

Suppressors and enhancers of expanded CUG toxicity affect nuclear foci. (A) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. Shown are 123CUG and the 0CUG control in different RNAi gene inactivations: empty vector control (ctrl), C06A1.6 and npp-4. Yellow arrows indicate expanded CUG nuclear foci. (B) Computational analysis of SM-FISH images of 123CUG animals with different gene inactivations and control (ctrl). Results are plotted as bar graphs were gene inactivations corresponding to bars on the right of the control exhibit an increase in detected foci area (in red), and conversely bars on the left of the control exhibit a decrease in foci area (in green), relative to the control. The cfim-2 and F48E8.6 gene inactivations are similar to ctrl. (C) C. elegans muscle cells confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue), merge of GFP RNA and nucleus images, and mCherry translational fusion protein. Strains imaged are 123CUG and 0CUG animals, in a mCHERRY (control) or NPP-4::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125460&req=5

Figure 3: Suppressors and enhancers of expanded CUG toxicity affect nuclear foci. (A) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. Shown are 123CUG and the 0CUG control in different RNAi gene inactivations: empty vector control (ctrl), C06A1.6 and npp-4. Yellow arrows indicate expanded CUG nuclear foci. (B) Computational analysis of SM-FISH images of 123CUG animals with different gene inactivations and control (ctrl). Results are plotted as bar graphs were gene inactivations corresponding to bars on the right of the control exhibit an increase in detected foci area (in red), and conversely bars on the left of the control exhibit a decrease in foci area (in green), relative to the control. The cfim-2 and F48E8.6 gene inactivations are similar to ctrl. (C) C. elegans muscle cells confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue), merge of GFP RNA and nucleus images, and mCherry translational fusion protein. Strains imaged are 123CUG and 0CUG animals, in a mCHERRY (control) or NPP-4::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci.

Mentions: We examined whether any of the 15 gene inactivations that modulated expanded CUG repeat toxicity changed RNA foci accumulation of 123CUG transcripts. One prediction is that gene inactivations that improve the motility of animals expressing 123CUG RNAs would also cause a decrease in foci size or number and similarly, gene inactivations that caused further motility impairment would lead to an increase in foci size or number (Table 1). Of the 15 genes identified, inactivation of ocrl-1/inositol-1,4,5-triphosphate 5-phosphatase, str-67/GPCR chemoreceptor and C06A1.6, led to an improvement of motility in strains expressing 123CUG repeats in muscle (Table 1). Examination of GFP mRNA localization by SM-FISH in 123CUG muscles revealed a significant reduction in the number of nuclear foci when these three genes are inactivated (Fig. 3A, B, Supplementary Fig. 4, 5). The suppression of 123CUG foci was particularly striking for C06A1.6 gene inactivation, where 123CUG foci were now few and small, with SM-FISH signals close to 0CUG control levels (Fig. 3A, Supplementary Fig. 4). However, distribution of expanded RNA ‘single’ transcripts was still observed preferentially in the nucleus versus the cytoplasm for all 3 gene inactivations, suggesting a role for ocrl-1, str-67 and C06A1.6 in foci formation rather than in cellular distribution of RNA. No significant changes in RNA localization, and no foci accumulation, were found in the control 0CUG strain, when these 3 genes were inactivated (Fig. 3A, Supplementary Fig. 4, 5). Together, these data support a model in which ocrl-1, str-67 and C06A1.6 gene activities normally enhance the toxicity of expanded CUG repeats by contributing to 123CUG foci formation, and inactivation of these genes results in decreased toxicity.


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)

Suppressors and enhancers of expanded CUG toxicity affect nuclear foci. (A) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. Shown are 123CUG and the 0CUG control in different RNAi gene inactivations: empty vector control (ctrl), C06A1.6 and npp-4. Yellow arrows indicate expanded CUG nuclear foci. (B) Computational analysis of SM-FISH images of 123CUG animals with different gene inactivations and control (ctrl). Results are plotted as bar graphs were gene inactivations corresponding to bars on the right of the control exhibit an increase in detected foci area (in red), and conversely bars on the left of the control exhibit a decrease in foci area (in green), relative to the control. The cfim-2 and F48E8.6 gene inactivations are similar to ctrl. (C) C. elegans muscle cells confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue), merge of GFP RNA and nucleus images, and mCherry translational fusion protein. Strains imaged are 123CUG and 0CUG animals, in a mCHERRY (control) or NPP-4::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125460&req=5

Figure 3: Suppressors and enhancers of expanded CUG toxicity affect nuclear foci. (A) Confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue) and merge of C. elegans muscle cells. Shown are 123CUG and the 0CUG control in different RNAi gene inactivations: empty vector control (ctrl), C06A1.6 and npp-4. Yellow arrows indicate expanded CUG nuclear foci. (B) Computational analysis of SM-FISH images of 123CUG animals with different gene inactivations and control (ctrl). Results are plotted as bar graphs were gene inactivations corresponding to bars on the right of the control exhibit an increase in detected foci area (in red), and conversely bars on the left of the control exhibit a decrease in foci area (in green), relative to the control. The cfim-2 and F48E8.6 gene inactivations are similar to ctrl. (C) C. elegans muscle cells confocal SM-FISH images of GFP RNA transcripts (white), DAPI stained nucleus (blue), merge of GFP RNA and nucleus images, and mCherry translational fusion protein. Strains imaged are 123CUG and 0CUG animals, in a mCHERRY (control) or NPP-4::mCHERRY backgrounds. Yellow arrows indicate expanded CUG nuclear foci.
Mentions: We examined whether any of the 15 gene inactivations that modulated expanded CUG repeat toxicity changed RNA foci accumulation of 123CUG transcripts. One prediction is that gene inactivations that improve the motility of animals expressing 123CUG RNAs would also cause a decrease in foci size or number and similarly, gene inactivations that caused further motility impairment would lead to an increase in foci size or number (Table 1). Of the 15 genes identified, inactivation of ocrl-1/inositol-1,4,5-triphosphate 5-phosphatase, str-67/GPCR chemoreceptor and C06A1.6, led to an improvement of motility in strains expressing 123CUG repeats in muscle (Table 1). Examination of GFP mRNA localization by SM-FISH in 123CUG muscles revealed a significant reduction in the number of nuclear foci when these three genes are inactivated (Fig. 3A, B, Supplementary Fig. 4, 5). The suppression of 123CUG foci was particularly striking for C06A1.6 gene inactivation, where 123CUG foci were now few and small, with SM-FISH signals close to 0CUG control levels (Fig. 3A, Supplementary Fig. 4). However, distribution of expanded RNA ‘single’ transcripts was still observed preferentially in the nucleus versus the cytoplasm for all 3 gene inactivations, suggesting a role for ocrl-1, str-67 and C06A1.6 in foci formation rather than in cellular distribution of RNA. No significant changes in RNA localization, and no foci accumulation, were found in the control 0CUG strain, when these 3 genes were inactivated (Fig. 3A, Supplementary Fig. 4, 5). Together, these data support a model in which ocrl-1, str-67 and C06A1.6 gene activities normally enhance the toxicity of expanded CUG repeats by contributing to 123CUG foci formation, and inactivation of these genes results in decreased toxicity.

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