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Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Le Mée G, Ezzeddine N, Capri M, Aït-Ahmed O - PLoS ONE (2008)

Bottom Line: In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene.Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci.This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique Humaine, Unité Propre de Recherche 1142, Centre National de la Recherche Scientifique, Montpellier, France.

ABSTRACT
Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

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(CTG)240.4 expansion and CG9650 target gene expression.A. CG9650 gene organization and insertion mapping. Three CG9650 mRNAs have been reported, form A is an EST, B and C are computationally derived. Lines represent introns and exons are represented as grey boxes. The (CTG)240.4 transgene (green) is inserted in the first intron of CG9650 at nucleotide 2051. The position of CG9650-rhodamin probes is shown on the diagram as red stars. B. RNA-FISH on salivary glands with CAG-FITC and CG9650-rhodamin probes (rho-CG96-1 and rho-CG96-2). Nuclei were counterstained with DAPI. CG9650 RNA forms nuclear foci that co-localize with (CUG)240.4 RNA. MS1096-Gal4 was used for induction. No FISH staining was detected when the transgene was not induced.
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pone-0001466-g006: (CTG)240.4 expansion and CG9650 target gene expression.A. CG9650 gene organization and insertion mapping. Three CG9650 mRNAs have been reported, form A is an EST, B and C are computationally derived. Lines represent introns and exons are represented as grey boxes. The (CTG)240.4 transgene (green) is inserted in the first intron of CG9650 at nucleotide 2051. The position of CG9650-rhodamin probes is shown on the diagram as red stars. B. RNA-FISH on salivary glands with CAG-FITC and CG9650-rhodamin probes (rho-CG96-1 and rho-CG96-2). Nuclei were counterstained with DAPI. CG9650 RNA forms nuclear foci that co-localize with (CUG)240.4 RNA. MS1096-Gal4 was used for induction. No FISH staining was detected when the transgene was not induced.

Mentions: We then investigated whether the toxic effect of the (CUG)240.4 RNA could be accounted for by the transgene insertion context. We assessed this hypothesis by performing inverse PCR amplification on genomic DNA prepared from the different transgenic lines. All but three lines had the transgene inserted in a non transcribed region of the genome (Table 1). The (CTG)240.4 line was one of the three exceptions. (CTG)240.4 insertion lies within the first intron of the CG9650 gene that encodes a zinc finger protein [20], [21], in the same orientation as the endogenous transcription unit (Figure 6A). To gain further understanding, we investigated the expression pattern of CG9650 in the (CTG)240.4 line after induction of the transgene in the salivary glands using the MS1096-Gal4 driver [24]. We performed RNA-FISH with CAG-FITC and CG9650-rhodamin probes (Figure 6A). We found that CG9650 RNA (as revealed with a probe that is complementary to exonic sequences) was concentrated in the same nuclear foci as the (CUG)240.4 RNA (Figure 6B). We did not observe formation of nuclear foci when CG9650 RNA was over-expressed in a context without any repeat expansion or when the RNA expansion was expressed in trans from an insertion that is located at a different locus (data not shown). Therefore, the (CUG)240.4/CG9650 foci could form only when CG9650 RNA and the CUG expansion lie in cis within the same transcript.


Repeat length and RNA expression level are not primary determinants in CUG expansion toxicity in Drosophila models.

Le Mée G, Ezzeddine N, Capri M, Aït-Ahmed O - PLoS ONE (2008)

(CTG)240.4 expansion and CG9650 target gene expression.A. CG9650 gene organization and insertion mapping. Three CG9650 mRNAs have been reported, form A is an EST, B and C are computationally derived. Lines represent introns and exons are represented as grey boxes. The (CTG)240.4 transgene (green) is inserted in the first intron of CG9650 at nucleotide 2051. The position of CG9650-rhodamin probes is shown on the diagram as red stars. B. RNA-FISH on salivary glands with CAG-FITC and CG9650-rhodamin probes (rho-CG96-1 and rho-CG96-2). Nuclei were counterstained with DAPI. CG9650 RNA forms nuclear foci that co-localize with (CUG)240.4 RNA. MS1096-Gal4 was used for induction. No FISH staining was detected when the transgene was not induced.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2186381&req=5

pone-0001466-g006: (CTG)240.4 expansion and CG9650 target gene expression.A. CG9650 gene organization and insertion mapping. Three CG9650 mRNAs have been reported, form A is an EST, B and C are computationally derived. Lines represent introns and exons are represented as grey boxes. The (CTG)240.4 transgene (green) is inserted in the first intron of CG9650 at nucleotide 2051. The position of CG9650-rhodamin probes is shown on the diagram as red stars. B. RNA-FISH on salivary glands with CAG-FITC and CG9650-rhodamin probes (rho-CG96-1 and rho-CG96-2). Nuclei were counterstained with DAPI. CG9650 RNA forms nuclear foci that co-localize with (CUG)240.4 RNA. MS1096-Gal4 was used for induction. No FISH staining was detected when the transgene was not induced.
Mentions: We then investigated whether the toxic effect of the (CUG)240.4 RNA could be accounted for by the transgene insertion context. We assessed this hypothesis by performing inverse PCR amplification on genomic DNA prepared from the different transgenic lines. All but three lines had the transgene inserted in a non transcribed region of the genome (Table 1). The (CTG)240.4 line was one of the three exceptions. (CTG)240.4 insertion lies within the first intron of the CG9650 gene that encodes a zinc finger protein [20], [21], in the same orientation as the endogenous transcription unit (Figure 6A). To gain further understanding, we investigated the expression pattern of CG9650 in the (CTG)240.4 line after induction of the transgene in the salivary glands using the MS1096-Gal4 driver [24]. We performed RNA-FISH with CAG-FITC and CG9650-rhodamin probes (Figure 6A). We found that CG9650 RNA (as revealed with a probe that is complementary to exonic sequences) was concentrated in the same nuclear foci as the (CUG)240.4 RNA (Figure 6B). We did not observe formation of nuclear foci when CG9650 RNA was over-expressed in a context without any repeat expansion or when the RNA expansion was expressed in trans from an insertion that is located at a different locus (data not shown). Therefore, the (CUG)240.4/CG9650 foci could form only when CG9650 RNA and the CUG expansion lie in cis within the same transcript.

Bottom Line: In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene.Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci.This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique Humaine, Unité Propre de Recherche 1142, Centre National de la Recherche Scientifique, Montpellier, France.

ABSTRACT
Evidence for an RNA gain-of-function toxicity has now been provided for an increasing number of human pathologies. Myotonic dystrophies (DM) belong to a class of RNA-dominant diseases that result from RNA repeat expansion toxicity. Specifically, DM of type 1 (DM1), is caused by an expansion of CUG repeats in the 3'UTR of the DMPK protein kinase mRNA, while DM of type 2 (DM2) is linked to an expansion of CCUG repeats in an intron of the ZNF9 transcript (ZNF9 encodes a zinc finger protein). In both pathologies the mutant RNA forms nuclear foci. The mechanisms that underlie the RNA pathogenicity seem to be rather complex and not yet completely understood. Here, we describe Drosophila models that might help unravelling the molecular mechanisms of DM1-associated CUG expansion toxicity. We generated transgenic flies that express inducible repeats of different type (CUG or CAG) and length (16, 240, 480 repeats) and then analyzed transgene localization, RNA expression and toxicity as assessed by induced lethality and eye neurodegeneration. The only line that expressed a toxic RNA has a (CTG)(240) insertion. Moreover our analysis shows that its level of expression cannot account for its toxicity. In this line, (CTG)(240.4), the expansion inserted in the first intron of CG9650, a zinc finger protein encoding gene. Interestingly, CG9650 and (CUG)(240.4) expansion RNAs were found in the same nuclear foci. In conclusion, we suggest that the insertion context is the primary determinant for expansion toxicity in Drosophila models. This finding should contribute to the still open debate on the role of the expansions per se in Drosophila and in human pathogenesis of RNA-dominant diseases.

Show MeSH
Related in: MedlinePlus