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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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Deregulation of the mitochondrial pathway in the OPMD mouse model.A) Number of deregulated genes in A17.1 mouse skeletal muscles using microarrays. T1, 6 weeks; T2, 18 weeks; T3, 26 weeks. B) Venn diagram of overlapping down-regulated genes at T1, T2 and T3. C) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in A17.1 mouse muscles. D) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control (WT) and A17.1 quadriceps skeletal muscle at T1, using RT-qPCR. Normalization was with Rplp0 mRNA. Means are from three biological replicates, error bars represent standard deviation. * p-value <0.05, ** p-value <0.01, *** p-value <0.001, using the Student’s t-Test. E) ePAT assays of mRNAs encoding mitochondrial proteins in control (WT) and A17.1 quadriceps skeletal muscles. Arrows indicate poly(A) tails of 12A. Accumulation of 12A poly(A) tails was visible in A17.1 muscles at T2 and/or T3. RpL32 is a negative control mRNA encoding a ribosomal protein. Profiles of ePAT assays using the ImageJ software are shown.
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pgen.1005092.g007: Deregulation of the mitochondrial pathway in the OPMD mouse model.A) Number of deregulated genes in A17.1 mouse skeletal muscles using microarrays. T1, 6 weeks; T2, 18 weeks; T3, 26 weeks. B) Venn diagram of overlapping down-regulated genes at T1, T2 and T3. C) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in A17.1 mouse muscles. D) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control (WT) and A17.1 quadriceps skeletal muscle at T1, using RT-qPCR. Normalization was with Rplp0 mRNA. Means are from three biological replicates, error bars represent standard deviation. * p-value <0.05, ** p-value <0.01, *** p-value <0.001, using the Student’s t-Test. E) ePAT assays of mRNAs encoding mitochondrial proteins in control (WT) and A17.1 quadriceps skeletal muscles. Arrows indicate poly(A) tails of 12A. Accumulation of 12A poly(A) tails was visible in A17.1 muscles at T2 and/or T3. RpL32 is a negative control mRNA encoding a ribosomal protein. Profiles of ePAT assays using the ImageJ software are shown.

Mentions: To extend our study to a mammalian model, quadriceps gene expression using microarrays was compared between control mouse (FvB) and A17.1 mouse which expresses PABPN1-17ala in skeletal muscle [53], at three time points (T1, 6 weeks; T2, 18 weeks; and T3, 26 weeks) [54]. Up- and down-regulated genes were found at all time points (Fig. 7A), with the ubiquitin-proteasome system being higly deregulated [9]. Annotation clustering enrichment analysis using DAVID [55] revealed that down-regulated genes common to all three time points were mostly enriched in genes encoding mitochondrial proteins (GO:0005739 "mitochondrion", Fold enrichment 14.8, p-value 5.88E-23), and we identified several nuclear genes involved in oxidative phosphorylation that were down-regulated, starting at the earliest time point (Fig. 7B, C, S3 Table). RT-qPCR confirmed the down-regulation observed with microarrays at T1 (Fig. 7D). These data obtained on pre-symptomatic muscles, for which no muscle weakness was evidenced [53], further confirmed the down-regulation of mRNAs encoding mitochondrial proteins as an early defect in OPMD. Using ePAT (extension PAT) assays, we found that the down-regulation of these mRNAs correlated with the accumulation of shorter poly(A) tails (Fig. 7E).


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)

Deregulation of the mitochondrial pathway in the OPMD mouse model.A) Number of deregulated genes in A17.1 mouse skeletal muscles using microarrays. T1, 6 weeks; T2, 18 weeks; T3, 26 weeks. B) Venn diagram of overlapping down-regulated genes at T1, T2 and T3. C) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in A17.1 mouse muscles. D) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control (WT) and A17.1 quadriceps skeletal muscle at T1, using RT-qPCR. Normalization was with Rplp0 mRNA. Means are from three biological replicates, error bars represent standard deviation. * p-value <0.05, ** p-value <0.01, *** p-value <0.001, using the Student’s t-Test. E) ePAT assays of mRNAs encoding mitochondrial proteins in control (WT) and A17.1 quadriceps skeletal muscles. Arrows indicate poly(A) tails of 12A. Accumulation of 12A poly(A) tails was visible in A17.1 muscles at T2 and/or T3. RpL32 is a negative control mRNA encoding a ribosomal protein. Profiles of ePAT assays using the ImageJ software are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4376527&req=5

pgen.1005092.g007: Deregulation of the mitochondrial pathway in the OPMD mouse model.A) Number of deregulated genes in A17.1 mouse skeletal muscles using microarrays. T1, 6 weeks; T2, 18 weeks; T3, 26 weeks. B) Venn diagram of overlapping down-regulated genes at T1, T2 and T3. C) Number of nuclear genes encoding mitochondrial respiratory chain complex subunits down-regulated in A17.1 mouse muscles. D) Quantification of levels of mRNAs encoding mitochondrial respiratory chain subunits in control (WT) and A17.1 quadriceps skeletal muscle at T1, using RT-qPCR. Normalization was with Rplp0 mRNA. Means are from three biological replicates, error bars represent standard deviation. * p-value <0.05, ** p-value <0.01, *** p-value <0.001, using the Student’s t-Test. E) ePAT assays of mRNAs encoding mitochondrial proteins in control (WT) and A17.1 quadriceps skeletal muscles. Arrows indicate poly(A) tails of 12A. Accumulation of 12A poly(A) tails was visible in A17.1 muscles at T2 and/or T3. RpL32 is a negative control mRNA encoding a ribosomal protein. Profiles of ePAT assays using the ImageJ software are shown.
Mentions: To extend our study to a mammalian model, quadriceps gene expression using microarrays was compared between control mouse (FvB) and A17.1 mouse which expresses PABPN1-17ala in skeletal muscle [53], at three time points (T1, 6 weeks; T2, 18 weeks; and T3, 26 weeks) [54]. Up- and down-regulated genes were found at all time points (Fig. 7A), with the ubiquitin-proteasome system being higly deregulated [9]. Annotation clustering enrichment analysis using DAVID [55] revealed that down-regulated genes common to all three time points were mostly enriched in genes encoding mitochondrial proteins (GO:0005739 "mitochondrion", Fold enrichment 14.8, p-value 5.88E-23), and we identified several nuclear genes involved in oxidative phosphorylation that were down-regulated, starting at the earliest time point (Fig. 7B, C, S3 Table). RT-qPCR confirmed the down-regulation observed with microarrays at T1 (Fig. 7D). These data obtained on pre-symptomatic muscles, for which no muscle weakness was evidenced [53], further confirmed the down-regulation of mRNAs encoding mitochondrial proteins as an early defect in OPMD. Using ePAT (extension PAT) assays, we found that the down-regulation of these mRNAs correlated with the accumulation of shorter poly(A) tails (Fig. 7E).

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