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Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis.

Chartier A, Klein P, Pierson S, Barbezier N, Gidaro T, Casas F, Carberry S, Dowling P, Maynadier L, Bellec M, Oloko M, Jardel C, Moritz B, Dickson G, Mouly V, Ohlendieck K, Butler-Browne G, Trollet C, Simonelig M - PLoS Genet. (2015)

Bottom Line: The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function.This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction.These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: mRNA Regulation and Development, Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France.

ABSTRACT
Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

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Reduced mitochondrial activity in muscles expressing PABPN1-17ala.A) Quantification of mitochondrial DNA (mtDNA) content in control and PABPN1-17ala- expressing thoraxes at day 2, using qPCR. Three mitochondrial genes (mt:CoI, mt:CoII and mt:cyt-b) were analysed. Mitochondrial DNA levels were normalized to RpL32 DNA. Means are from three biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test. B) Activities of mitochondrial respiratory chain complexes were analysed by spectrophotometry from control and PABPN1-17ala-expressing thoraxes (genotypes as in A). Means are from five biological replicates, error bars represent standard deviation. * p-value <0.05, *** p-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of mRNA levels of transcription factors regulating mitochondrial function in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR (genotypes as in A). mRNA levels were normalized to sop mRNA. Means are from two biological replicates quantified three times, error bars represent standard deviation. *** p-value <0.001, ns: not significant, using the Student’s t-Test. D) Overexpression of ewg and dERR genes reduces the wing posture phenotypes of flies expressing PABPN1-17ala. Wing posture phenotypes were scored at day 6, at 18°C from OPMD (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/+), OPMD; OE-spargel (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/spargelEY05931), OPMD; OE-delg (w1118; UAS-PABPN1-17ala, UAS-delg-HA/+; Mhc-Gal4/+), OPMD; OE-ewg (w, ewgEY05137/Y; UAS-PABPN1-17ala/+; Mhc-Gal4/+) and OPMD; OE-dERR (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/dERRG4389) flies (n > 130). *** p-value <0.001, using the χ2 test.
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pgen.1005092.g002: Reduced mitochondrial activity in muscles expressing PABPN1-17ala.A) Quantification of mitochondrial DNA (mtDNA) content in control and PABPN1-17ala- expressing thoraxes at day 2, using qPCR. Three mitochondrial genes (mt:CoI, mt:CoII and mt:cyt-b) were analysed. Mitochondrial DNA levels were normalized to RpL32 DNA. Means are from three biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test. B) Activities of mitochondrial respiratory chain complexes were analysed by spectrophotometry from control and PABPN1-17ala-expressing thoraxes (genotypes as in A). Means are from five biological replicates, error bars represent standard deviation. * p-value <0.05, *** p-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of mRNA levels of transcription factors regulating mitochondrial function in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR (genotypes as in A). mRNA levels were normalized to sop mRNA. Means are from two biological replicates quantified three times, error bars represent standard deviation. *** p-value <0.001, ns: not significant, using the Student’s t-Test. D) Overexpression of ewg and dERR genes reduces the wing posture phenotypes of flies expressing PABPN1-17ala. Wing posture phenotypes were scored at day 6, at 18°C from OPMD (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/+), OPMD; OE-spargel (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/spargelEY05931), OPMD; OE-delg (w1118; UAS-PABPN1-17ala, UAS-delg-HA/+; Mhc-Gal4/+), OPMD; OE-ewg (w, ewgEY05137/Y; UAS-PABPN1-17ala/+; Mhc-Gal4/+) and OPMD; OE-dERR (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/dERRG4389) flies (n > 130). *** p-value <0.001, using the χ2 test.

Mentions: Because down-regulation of mitochondrial components is prominent at the earliest time point, we checked whether mitochondrial biogenesis or mass were affected in muscles expressing PABPN1-17ala. Mitochondrial abundance was analysed by quantifying mitochondrial DNA using qPCR. Mitochondrial DNA levels normalized to nuclear DNA were unaffected in Drosophila muscles expressing PABPN1-17ala, suggesting that mitochondrial mass was similar to that in control muscles (Fig. 2A).


Mitochondrial dysfunction reveals the role of mRNA poly(A) tail regulation in oculopharyngeal muscular dystrophy pathogenesis.

Chartier A, Klein P, Pierson S, Barbezier N, Gidaro T, Casas F, Carberry S, Dowling P, Maynadier L, Bellec M, Oloko M, Jardel C, Moritz B, Dickson G, Mouly V, Ohlendieck K, Butler-Browne G, Trollet C, Simonelig M - PLoS Genet. (2015)

Reduced mitochondrial activity in muscles expressing PABPN1-17ala.A) Quantification of mitochondrial DNA (mtDNA) content in control and PABPN1-17ala- expressing thoraxes at day 2, using qPCR. Three mitochondrial genes (mt:CoI, mt:CoII and mt:cyt-b) were analysed. Mitochondrial DNA levels were normalized to RpL32 DNA. Means are from three biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test. B) Activities of mitochondrial respiratory chain complexes were analysed by spectrophotometry from control and PABPN1-17ala-expressing thoraxes (genotypes as in A). Means are from five biological replicates, error bars represent standard deviation. * p-value <0.05, *** p-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of mRNA levels of transcription factors regulating mitochondrial function in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR (genotypes as in A). mRNA levels were normalized to sop mRNA. Means are from two biological replicates quantified three times, error bars represent standard deviation. *** p-value <0.001, ns: not significant, using the Student’s t-Test. D) Overexpression of ewg and dERR genes reduces the wing posture phenotypes of flies expressing PABPN1-17ala. Wing posture phenotypes were scored at day 6, at 18°C from OPMD (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/+), OPMD; OE-spargel (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/spargelEY05931), OPMD; OE-delg (w1118; UAS-PABPN1-17ala, UAS-delg-HA/+; Mhc-Gal4/+), OPMD; OE-ewg (w, ewgEY05137/Y; UAS-PABPN1-17ala/+; Mhc-Gal4/+) and OPMD; OE-dERR (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/dERRG4389) flies (n > 130). *** p-value <0.001, using the χ2 test.
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pgen.1005092.g002: Reduced mitochondrial activity in muscles expressing PABPN1-17ala.A) Quantification of mitochondrial DNA (mtDNA) content in control and PABPN1-17ala- expressing thoraxes at day 2, using qPCR. Three mitochondrial genes (mt:CoI, mt:CoII and mt:cyt-b) were analysed. Mitochondrial DNA levels were normalized to RpL32 DNA. Means are from three biological replicates, error bars represent standard deviation. ns: not significant, using the Student’s t-Test. B) Activities of mitochondrial respiratory chain complexes were analysed by spectrophotometry from control and PABPN1-17ala-expressing thoraxes (genotypes as in A). Means are from five biological replicates, error bars represent standard deviation. * p-value <0.05, *** p-value <0.001, ns: not significant, using the Student’s t-Test. C) Quantification of mRNA levels of transcription factors regulating mitochondrial function in control and PABPN1-17ala-expressing thoraxes at day 2, using RT-qPCR (genotypes as in A). mRNA levels were normalized to sop mRNA. Means are from two biological replicates quantified three times, error bars represent standard deviation. *** p-value <0.001, ns: not significant, using the Student’s t-Test. D) Overexpression of ewg and dERR genes reduces the wing posture phenotypes of flies expressing PABPN1-17ala. Wing posture phenotypes were scored at day 6, at 18°C from OPMD (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/+), OPMD; OE-spargel (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/spargelEY05931), OPMD; OE-delg (w1118; UAS-PABPN1-17ala, UAS-delg-HA/+; Mhc-Gal4/+), OPMD; OE-ewg (w, ewgEY05137/Y; UAS-PABPN1-17ala/+; Mhc-Gal4/+) and OPMD; OE-dERR (w1118; UAS-PABPN1-17ala/+; Mhc-Gal4/dERRG4389) flies (n > 130). *** p-value <0.001, using the χ2 test.
Mentions: Because down-regulation of mitochondrial components is prominent at the earliest time point, we checked whether mitochondrial biogenesis or mass were affected in muscles expressing PABPN1-17ala. Mitochondrial abundance was analysed by quantifying mitochondrial DNA using qPCR. Mitochondrial DNA levels normalized to nuclear DNA were unaffected in Drosophila muscles expressing PABPN1-17ala, suggesting that mitochondrial mass was similar to that in control muscles (Fig. 2A).

Bottom Line: The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function.This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction.These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

View Article: PubMed Central - PubMed

Affiliation: mRNA Regulation and Development, Institut de Génétique Humaine, CNRS UPR1142, Montpellier, France.

ABSTRACT
Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.

Show MeSH
Related in: MedlinePlus