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Anita H. Corbett - One of the best experts on this subject based on the ideXlab platform.
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Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle
The Journal of biological chemistry, 2019Co-Authors: Ayan Banerjee, Katherine E. Vest, Brittany L. Phillips, Grace K. Pavlath, Quidong Deng, Nicholas T. Seyfried, Anita H. CorbettAbstract:Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.
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Post-transcriptional regulation of PABPN1 by the RNA binding protein HuR
Nucleic acids research, 2018Co-Authors: Brittany L. Phillips, Ayan Banerjee, Grace K. Pavlath, Brenda Janice Sanchez, Sergio Marco, Imed-eddine Gallouzi, Anita H. CorbettAbstract:RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the PABPN1 transcript is unstable, contributing to low steady-state PABPN1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate PABPN1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate PABPN1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases PABPN1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates PABPN1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.
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Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing.
Nucleic acids research, 2017Co-Authors: Ayan Banerjee, Katherine E. Vest, Grace K. Pavlath, Anita H. CorbettAbstract:The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle. We have used co-immunoprecipitation and mass spectrometry to identify proteins that interact with PABPN1 in mouse skeletal muscles. Among the interacting proteins we identified Matrin 3 (MATR3) as a novel protein interactor of PABPN1. The MATR3 gene is mutated in a form of distal myopathy and amyotrophic lateral sclerosis (ALS). We demonstrate, that like PABPN1, MATR3 is critical for myogenesis. Furthermore, MATR3 controls critical aspects of RNA processing including alternative polyadenylation and intron retention. We provide evidence that MATR3 also binds and regulates the levels of long non-coding RNA (lncRNA) Neat1 and together with PABPN1 is required for normal paraspeckle function. We demonstrate that PABPN1 and MATR3 are required for paraspeckles, as well as for adenosine to inosine (A to I) RNA editing of Ctn RNA in muscle cells. We provide a functional link between PABPN1 and MATR3 through regulation of a common lncRNA target with downstream impact on paraspeckle morphology and function. We extend our analysis to a mouse model of OPMD and demonstrate altered paraspeckle morphology in the presence of endogenous levels of alanine-expanded PABPN1. In this study, we report protein-binding partners of PABPN1, which could provide insight into novel functions of PABPN1 in skeletal muscle and identify proteins that could be sequestered with alanine-expanded PABPN1 in the nuclear aggregates found in OPMD.
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An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.
Journal of neuromuscular diseases, 2015Co-Authors: Katherine E. Vest, Ayan Banerjee, Luciano H. Apponi, Grace K. Pavlath, Anita H. CorbettAbstract:Background Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs. Objective We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1. Methods We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD. Results The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD. Conclusions This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.
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Control of mRNA stability contributes to low levels of nuclear poly(A) binding protein 1 (PABPN1) in skeletal muscle.
Skeletal muscle, 2013Co-Authors: Luciano H. Apponi, Anita H. Corbett, Grace K. PavlathAbstract:The nuclear poly(A) binding protein 1 (PABPN1) is a ubiquitously expressed proteinthat plays critical roles at multiple steps in post-transcriptional regulation ofgene expression. Short expansions of the polyalanine tract in the N-terminus ofPABPN1 lead to oculopharyngeal muscular dystrophy (OPMD), which is an adult onsetdisease characterized by eyelid drooping, difficulty in swallowing, and weaknessin the proximal limb muscles. Why alanine-expanded PABPN1 leads to muscle-specificpathology is unknown. Given the general function of PABPN1 in RNA metabolism,intrinsic characteristics of skeletal muscle may make this tissue susceptible tothe effects of mutant PABPN1. To begin to understand the muscle specificity of OPMD, we investigated thesteady-state levels of PABPN1 in different tissues of humans and mice.Additionally, we analyzed the levels of PABPN1 during muscle regeneration afterinjury in mice. Furthermore, we assessed the dynamics of PABPN1 mRNA decay inskeletal muscle compared to kidney. Here, we show that the steady-state levels of both PABPN1 mRNA and protein aredrastically lower in mouse and human skeletal muscle, particularly those impactedin OPMD, compared to other tissues. In contrast, PABPN1 levels are increasedduring muscle regeneration, suggesting a greater requirement for PABPN1 functionduring tissue repair. Further analysis indicates that modulation of PABPN1expression is likely due to post-transcriptional mechanisms acting at the level ofmRNA stability. Our results demonstrate that PABPN1 steady-state levels and likely control ofexpression differ significantly in skeletal muscle as compared to other tissues,which could have important implications for understanding the muscle-specificnature of OPMD.
Guy A. Rouleau - One of the best experts on this subject based on the ideXlab platform.
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Cross-talk between canonical Wnt signaling and the sirtuin-FoxO longevity pathway to protect against muscular pathology induced by mutant PABPN1 expression in C. elegans.
Neurobiology of disease, 2010Co-Authors: Matthieu Y. Pasco, Bernard Brais, Guy A. Rouleau, Hélène Catoire, J. Alex Parker, Christian NeriAbstract:Abstract Developmental pathways may be play a role in adult cell survival. However, whether they interact with longevity/cell survival pathways to confer protection against disease-associated proteotoxicity remains largely unknown. We previously reported that the inhibition of key longevity modulators such as the deacetylase sir-2.1 /SIRT1 (Sir2) and its target daf-16 /FoxO protects transgenics nematodes from muscle cell decline and abnormal motility produced by the expression of mutant (polyalanine-expanded) PABPN1, the oculopharyngeal muscular dystrophy (OPMD) protein. Here, we report that canonical Wnt signaling ( i ) modulates muscular pathology in mutant PABPN1 nematodes, and ( ii ) cooperates with the Sir2-FoxO longevity pathway to confer protection against mutant PABPN1 toxicity at the cellular and behavioral levels. Mutant PABPN1 toxicity was modified by genes along the canonical Wnt pathway, several of which depend on daf-16 for activity. s-catenin and pop-1 /TCF RNAi suppressed the protection from mutant PABPN1 confered by loss-of-function mutations in sir-2.1 and daf-16 . Moreover, the aggravation of muscle cell pathology by increased sir-2.1 dosage was reversed by s-catenin and pop-1 RNAi. The chemical inhibition of GSK-3s, a repressor of s-catenin activity, protected against mutant PABPN1 toxicity in a daf-16 -dependent manner, which is consistent with a cross-talk between s-catenin signaling and Sir2-FoxO signaling in protecting from mutant PABPN1 toxicity. Our data reveal that canonical Wnt signaling and Sir2-FoxO signaling interact to modulate diseased muscle survival, and indicate that GSK-3s inhibitors and sirtuin inhibitors both have therapeutic potential for muscle protection in OPMD.
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Characterization of Sirtuin Inhibitors in Nematodes Expressing a Muscular Dystrophy Protein Reveals Muscle Cell and Behavioral Protection by Specific Sirtinol Analogues
Journal of medicinal chemistry, 2010Co-Authors: Matthieu Y. Pasco, Guy A. Rouleau, Dante Rotili, Lucia Altucci, Francesca Farina, Antonello Mai, Christian NeriAbstract:In oculopharyngeal muscular dystrophy (OPMD), a disease caused by polyalanine expansion in the nuclear protein PABPN1, the genetic inhibition of sirtuins and treatment with sirtuin inhibitors protect from mutant PABPN1 toxicity in transgenic nematodes. Here, we tested the SIRT1/2 inhibitors 1−12, bearing different degrees of inhibition, for protection against mutant PABPN1 toxicity in Caenorhabditis elegans. Compounds 2, 4, and 11 were the most efficient, revealing a potential therapeutic application for muscle cell protection in OPMD.
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PABPN1 polyalanine tract deletion and long expansions modify its aggregation pattern and expression.
Experimental cell research, 2008Co-Authors: Arnaud F. Klein, Guy A. Rouleau, Mitsuru Ebihara, Christine Alexander, Marie-josée Dicaire, A. Marie-josée Sasseville, Yves Langelier, Bernard BraisAbstract:Expansions of a (GCN)10/polyalanine tract in the Poly(A) Binding Protein Nuclear 1 (PABPN1) cause autosomal dominant oculopharyngeal muscular dystrophy (OPMD). In OPMD muscles, as in models, PABPN1 accumulates in intranuclear inclusions (INIs) whereas in other diseases caused by similar polyalanine expansions, the mutated proteins have been shown to abnormally accumulate in the cytoplasm. This study presents the impact on the subcellular localization of PABPN1 produced by large expansions or deletion of its polyalanine tract. Large tracts of more than 24 alanines result in the nuclear accumulation of PABPN1 in SFRS2-positive functional speckles and a significant decline in cell survival. These large expansions do not cause INIs formation nor do they lead to cytoplasmic accumulation. Deletion of the polyalanine tract induces the formation of aggregates that are located on either side and cross the nuclear membrane, highlighting the possible role of the N-terminal polyalanine tract in PABPN1 nucleo-cytoplasmic transport. We also show that even though five other proteins with polyalanine tracts tend to aggregate when over-expressed they do not co-aggregate with PABPN1 INIs. This study presents the first experimental evidence that there may be a relative loss of function in OPMD by decreasing the availability of PABPN1 through an INI-independent mechanism.
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Sirtuin inhibition protects from the polyalanine muscular dystrophy protein PABPN1
Human Molecular Genetics, 2008Co-Authors: Hélène Catoire, Bernard Brais, Guy A. Rouleau, Aida Abu-baker, Matthieu Y. Pasco, J. Alex Parker, Sébastien Holbert, Cendrine Tourette, Christian NeriAbstract:Oculopharyngeal muscular dystrophy (OPMD) is caused by polyalanine expansion in nuclear protein PABPN1 [poly(A) binding protein nuclear 1] and characterized by muscle degeneration. Druggable modifiers of proteotoxicity in degenerative diseases, notably the longevity modulators sirtuins, may constitute useful therapeutic targets. However, the modifiers of mutant PABPN1 are unknown. Here, we report that longevity and cell metabolism modifiers modulate mutant PABPN1 toxicity in the muscle cell. Using PABPN1 nematodes that show muscle cell degeneration and abnormal motility, we found that increased dosage of the sirtuin and deacetylase sir-2.1/SIRT1 exacerbated muscle pathology, an effect dependent on the transcription factor daf-16/FoxO and fuel sensor aak-2/AMPK (AMP-activated protein kinase), while null mutants of sir-2.1, daf-16 and aak-2 were protective. Consistently, the Sir2 inhibitor sirtinol was protective, whereas the Sir2 and AMPK activator resveratrol was detrimental. Furthermore, rescue by sirtinol was dependent on daf-16 and not aak-2, whereas aggravation by resveratrol was dependent on aak-2 and not daf-16. Finally, the survival of mammalian cells expressing mutant PABPN1 was promoted by sirtinol and decreased by resveratrol. Altogether, our data identify Sir2 and AMPK inhibition as therapeutic strategies for muscle protection in OPMD, extending the value of druggable proteins in cell maintenance networks to polyalanine diseases.
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Oculopharyngeal muscular dystrophy: recent advances in the understanding of the molecular pathogenic mechanisms and treatment strategies.
Biochimica et biophysica acta, 2006Co-Authors: Aida Abu-baker, Guy A. RouleauAbstract:Oculopharyngeal muscular dystrophy (OPMD) is an adult-onset disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. OPMD is caused by a small expansion of a short polyalanine tract in the poly (A) binding protein nuclear 1 protein (PABPN1). The mechanism by which the polyalanine expansion mutation in PABPN1 causes disease is unclear. PABPN1 is a nuclear multi-functional protein which is involved in pre-mRNA polyadenylation, transcription regulation, and mRNA nucleocytoplasmic transport. The distinct pathological hallmark of OPMD is the presence of filamentous intranuclear inclusions (INIs) in patient's skeletal muscle cells. The exact relationship between mutant PABPN1 intranuclear aggregates and pathology is not clear. OPMD is a unique disease sharing common pathogenic features with other polyalanine disorders, as well as with polyglutamine and dystrophic disorders. This chapter aims to review the rapidly growing body of knowledge concerning OPMD. First, we outline the background of OPMD. Second, we compare OPMD with other trinucleotide repeat disorders. Third, we discuss the recent advances in the understanding of the molecular mechanisms underlying OPMD pathogenesis. Finally, we review recent therapeutic strategies for OPMD.
Grace K. Pavlath - One of the best experts on this subject based on the ideXlab platform.
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Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle
The Journal of biological chemistry, 2019Co-Authors: Ayan Banerjee, Katherine E. Vest, Brittany L. Phillips, Grace K. Pavlath, Quidong Deng, Nicholas T. Seyfried, Anita H. CorbettAbstract:Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.
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Post-transcriptional regulation of PABPN1 by the RNA binding protein HuR
Nucleic acids research, 2018Co-Authors: Brittany L. Phillips, Ayan Banerjee, Grace K. Pavlath, Brenda Janice Sanchez, Sergio Marco, Imed-eddine Gallouzi, Anita H. CorbettAbstract:RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the PABPN1 transcript is unstable, contributing to low steady-state PABPN1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate PABPN1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate PABPN1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases PABPN1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates PABPN1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.
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Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing.
Nucleic acids research, 2017Co-Authors: Ayan Banerjee, Katherine E. Vest, Grace K. Pavlath, Anita H. CorbettAbstract:The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle. We have used co-immunoprecipitation and mass spectrometry to identify proteins that interact with PABPN1 in mouse skeletal muscles. Among the interacting proteins we identified Matrin 3 (MATR3) as a novel protein interactor of PABPN1. The MATR3 gene is mutated in a form of distal myopathy and amyotrophic lateral sclerosis (ALS). We demonstrate, that like PABPN1, MATR3 is critical for myogenesis. Furthermore, MATR3 controls critical aspects of RNA processing including alternative polyadenylation and intron retention. We provide evidence that MATR3 also binds and regulates the levels of long non-coding RNA (lncRNA) Neat1 and together with PABPN1 is required for normal paraspeckle function. We demonstrate that PABPN1 and MATR3 are required for paraspeckles, as well as for adenosine to inosine (A to I) RNA editing of Ctn RNA in muscle cells. We provide a functional link between PABPN1 and MATR3 through regulation of a common lncRNA target with downstream impact on paraspeckle morphology and function. We extend our analysis to a mouse model of OPMD and demonstrate altered paraspeckle morphology in the presence of endogenous levels of alanine-expanded PABPN1. In this study, we report protein-binding partners of PABPN1, which could provide insight into novel functions of PABPN1 in skeletal muscle and identify proteins that could be sequestered with alanine-expanded PABPN1 in the nuclear aggregates found in OPMD.
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Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology
Human molecular genetics, 2017Co-Authors: Katherine E. Vest, Dinghai Zheng, Brittany L. Phillips, Ayan Banerjee, Luciano H. Apponi, Eric B. Dammer, Bin Tian, Grace K. PavlathAbstract:Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (PABPN1+/A17) that contains one alanine-expanded PABPN1 allele under the control of the native promoter and one wild-type PABPN1 allele. This mouse is the closest available genocopy of OPMD patients. We show that PABPN1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous PABPN1 knock-out mouse model (PABPN1+/Δ). Like the PABPN1+/A17 mice, PABPN1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in PABPN1+/Δ mice only partially overlap with those detected in PABPN1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in PABPN1+/A17 mice.
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An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.
Journal of neuromuscular diseases, 2015Co-Authors: Katherine E. Vest, Ayan Banerjee, Luciano H. Apponi, Grace K. Pavlath, Anita H. CorbettAbstract:Background Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs. Objective We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1. Methods We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD. Results The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD. Conclusions This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.
Vered Raz - One of the best experts on this subject based on the ideXlab platform.
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Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting
iScience, 2019Co-Authors: Cyriel Sebastiaan Olie, Muhammad Riaz, Annemieke Aartsma-rus, Jelle J Goeman, Rebecca Konietzny, Philip D. Charles, Adan Pinto-fernandez, Szymon M. Kielbasa, Benedikt M. Kessler, Vered RazAbstract:Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. VIDEO ABSTRACT.
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Deacetylation Inhibition Reverses PABPN1-Dependent Muscle Wasting
Elsevier, 2019Co-Authors: Cyriel Sebastiaan Olie, Muhammad Riaz, Jelle J Goeman, Rebecca Konietzny, Philip D. Charles, Adan Pinto-fernandez, Benedikt M. Kessler, Szymon M. Kiełbasa, A. Aartsma-rus, Vered RazAbstract:Summary: Reduced poly(A)-binding protein nuclear 1 (PABPN1) levels cause aging-associated muscle wasting. PABPN1 is a multifunctional regulator of mRNA processing. To elucidate the molecular mechanisms causing PABPN1-mediated muscle wasting, we compared the transcriptome with the proteome in mouse muscles expressing short hairpin RNA to PABPN1 (shPab). We found greater variations in the proteome than in mRNA expression profiles. Protein accumulation in the shPab proteome was concomitant with reduced proteasomal activity. Notably, protein acetylation appeared to be decreased in shPab versus control proteomes (63%). Acetylome profiling in shPab muscles revealed prominent peptide deacetylation associated with elevated sirtuin-1 (SIRT1) deacetylase. We show that SIRT1 mRNA levels are controlled by PABPN1 via alternative polyadenylation site utilization. Most importantly, SIRT1 deacetylase inhibition by sirtinol increased PABPN1 levels and reversed muscle wasting. We suggest that perturbation of a multifactorial regulatory loop involving PABPN1 and SIRT1 plays an imperative role in aging-associated muscle wasting. Video Abstract: : Biological Sciences; Physiology; Molecular Biology Subject Areas: Biological Sciences, Physiology, Molecular Biolog
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Dysfunctional transcripts are formed by alternative polyadenylation in OPMD
Oncotarget, 2017Co-Authors: Vered Raz, George Dickson, Peter A C T HoenAbstract:// Vered Raz 1 , George Dickson 2 and Peter A. C. ’t Hoen 1 1 Department of Human Genetics, Leiden University Medical Centre, Leiden, The Netherlands 2 School of Biological Science, Royal Holloway University of London, Egham, Surrey, United Kingdom Correspondence to: Vered Raz, email: // Keywords : PABPN1, mRNA processing, alternative polyadenylation site, autophagy, aging muscles, Gerotarget Received : April 11, 2017 Accepted : August 03, 2017 Published : September 05, 2017 Abstract Post-transcription mRNA processing in the 3’-untranslated region (UTR) of transcripts alters mRNA landscape. Alternative polyadenylation (APA) utilization in the 3’-UTR often leads to shorter 3’-UTR affecting mRNA stability, a process that is regulated by PABPN1. In skeletal muscles PABPN1 levels reduce with age and a greater decrease in found in Oculopharyngeal muscular dystrophy (OPMD). OPMD is a late onset autosomal dominant myopathy caused by expansion mutation in PABPN1. In OPMD models a shift from distal to proximal polyadenylation site utilization in the 3’-UTR, and PABPN1 was shown to play a prominent role in APA. Whether PABPN1-mediated APA transcripts are functional is not fully understood. We investigate nuclear export and translation efficiency of transcripts in OPMD models. We focused on autophagy-regulated genes (ATGs) with APA utilization in cell models with reduced functional PABPN1. We provide evidence that ATGs transcripts from distal PAS retain in the nucleus and thus have reduced translation efficiency in cells with reduced PABPN1. In contrast, transcripts from proximal PAS showed a higher cytoplasmic abundance but a reduced occupancy in the ribosome. We therefore suggest that in reduced PABPN1 levels ATG transcripts from APA may not effectively translate to proteins. In those conditions we found constitutive autophagosome fusion and reduced autophagy flux. Augmentation of PABPN1 restored autophagosome fusion, suggesting that PABPN1-mediated APA plays a role in autophagy in OPMD and in aging muscles.
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PABPN1-Dependent mRNA Processing Induces Muscle Wasting.
PLoS genetics, 2016Co-Authors: Muhammad Riaz, Yotam Raz, Maaike Van Putten, Guillem Paniagua-soriano, Yvonne D. Krom, Bogdan I. Florea, Vered RazAbstract:Poly(A) Binding Protein Nuclear 1 (PABPN1) is a multifunctional regulator of mRNA processing, and its expression levels specifically decline in aging muscles. An expansion mutation in PABPN1 is the genetic cause of oculopharyngeal muscle dystrophy (OPMD), a late onset and rare myopathy. Moreover, reduced PABPN1 expression correlates with symptom manifestation in OPMD. PABPN1 regulates alternative polyadenylation site (PAS) utilization. However, the impact of PAS utilization on cell and tissue function is poorly understood. We hypothesized that altered PABPN1 expression levels is an underlying cause of muscle wasting. To test this, we stably down-regulated PABPN1 in mouse tibialis anterior (TA) muscles by localized injection of adeno-associated viruses expressing shRNA to PABPN1 (shPab). We found that a mild reduction in PABPN1 levels causes muscle pathology including myofiber atrophy, thickening of extracellular matrix and myofiber-type transition. Moreover, reduced PABPN1 levels caused a consistent decline in distal PAS utilization in the 3’-UTR of a subset of OPMD-dysregulated genes. This alternative PAS utilization led to up-regulation of Atrogin-1, a key muscle atrophy regulator, but down regulation of proteasomal genes. Additionally reduced PABPN1 levels caused a reduction in proteasomal activity, and transition in MyHC isotope expression pattern in myofibers. We suggest that PABPN1-mediated alternative PAS utilization plays a central role in aging-associated muscle wasting.
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PABPN1-DR in mouse TA muscles.
2016Co-Authors: Muhammad Riaz, Yotam Raz, Maaike Van Putten, Guillem Paniagua-soriano, Yvonne D. Krom, Bogdan I. Florea, Vered RazAbstract:A. A representative fluorescence image at four weeks post-injection of AAV containing shRNA construct to PABPN1 or Scramble RNA. The GFP signal is localized into the injected TA muscles. B. GFP expression is stabilized three weeks post injection. C. Box plots show fold-change of eGFP mRNA expression in five mice and averages were normalized to PBS injected mice. D-F. PABPN1 levels. D. Dot plot shows PABPN1 mRNA Hprt and Gapdh levels in all injected mice. Hprt and Gapdh levels were normalized to mean Ct value in PBS injected mice. PABPN1 levels were normalization to the mean of Gapdh and Hprt and PBS injected. Faded color circles mark mice with lower PABPN1 levels. Means and SD are depicted with lines. E. A representative Western blot shows PABPN1 and Tubulin proteins. Loading controls are denoted by Tubulin or Coomassie blue (CB) stained gel. F. Bar chart shows mean PABPN1 accumulation is Scram and shPab muscles. PABPN1 levels were normalized to the average of three proteins (45, 47 and 51 kDa) from the CB gel. Averages and SD are from three mice with lowest PABPN1 levels. Statistical significance was assessed by paired Student’s t-tests. P-values
Ayan Banerjee - One of the best experts on this subject based on the ideXlab platform.
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Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle
The Journal of biological chemistry, 2019Co-Authors: Ayan Banerjee, Katherine E. Vest, Brittany L. Phillips, Grace K. Pavlath, Quidong Deng, Nicholas T. Seyfried, Anita H. CorbettAbstract:Oculopharyngeal muscular dystrophy (OPMD) is a late-onset, primarily autosomal dominant disease caused by a short GCN expansion in the PABPN1 (polyadenylate-binding protein nuclear 1) gene that results in an alanine expansion at the N terminus of the PABPN1 protein. Expression of alanine-expanded PABPN1 is linked to the formation of nuclear aggregates in tissues from individuals with OPMD. However, as with other nuclear aggregate-associated diseases, controversy exists over whether these aggregates are the direct cause of pathology. An emerging hypothesis is that a loss of PABPN1 function and/or aberrant protein interactions contribute to pathology in OPMD. Here, we present the first global proteomic analysis of the protein interactions of WT and alanine-expanded PABPN1 in skeletal muscle tissue. These data provide both insight into the function of PABPN1 in muscle and evidence that the alanine expansion alters the protein-protein interactions of PABPN1. We extended this analysis to demonstrate altered complex formation with and loss of function of TDP-43 (TAR DNA-binding protein 43), which we show interacts with alanine-expanded but not WT PABPN1. The results from our study support a model where altered protein interactions with alanine-expanded PABPN1 that lead to loss or gain of function could contribute to pathology in OPMD.
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Post-transcriptional regulation of PABPN1 by the RNA binding protein HuR
Nucleic acids research, 2018Co-Authors: Brittany L. Phillips, Ayan Banerjee, Grace K. Pavlath, Brenda Janice Sanchez, Sergio Marco, Imed-eddine Gallouzi, Anita H. CorbettAbstract:RNA processing is critical for proper spatial and temporal control of gene expression. The ubiquitous nuclear polyadenosine RNA binding protein, PABPN1, post-transcriptionally regulates multiple steps of gene expression. Mutations in the PABPN1 gene expanding an N-terminal alanine tract in the PABPN1 protein from 10 alanines to 11-18 alanines cause the muscle-specific disease oculopharyngeal muscular dystrophy (OPMD), which affects eyelid, pharynx, and proximal limb muscles. Previous work revealed that the PABPN1 transcript is unstable, contributing to low steady-state PABPN1 mRNA and protein levels in vivo, specifically in skeletal muscle, with even lower levels in muscles affected in OPMD. Thus, low levels of PABPN1 protein could predispose specific tissues to pathology in OPMD. However, no studies have defined the mechanisms that regulate PABPN1 expression. Here, we define multiple cis-regulatory elements and a trans-acting factor, HuR, which regulate PABPN1 expression specifically in mature muscle in vitro and in vivo. We exploit multiple models including C2C12 myotubes, primary muscle cells, and mice to determine that HuR decreases PABPN1 expression. Overall, we have uncovered a mechanism in mature muscle that negatively regulates PABPN1 expression in vitro and in vivo, which could provide insight to future studies investigating therapeutic strategies for OPMD treatment.
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Nuclear poly(A) binding protein 1 (PABPN1) and Matrin3 interact in muscle cells and regulate RNA processing.
Nucleic acids research, 2017Co-Authors: Ayan Banerjee, Katherine E. Vest, Grace K. Pavlath, Anita H. CorbettAbstract:The polyadenylate binding protein 1 (PABPN1) is a ubiquitously expressed RNA binding protein vital for multiple steps in RNA metabolism. Although PABPN1 plays a critical role in the regulation of RNA processing, mutation of the gene encoding this ubiquitously expressed RNA binding protein causes a specific form of muscular dystrophy termed oculopharyngeal muscular dystrophy (OPMD). Despite the tissue-specific pathology that occurs in this disease, only recently have studies of PABPN1 begun to explore the role of this protein in skeletal muscle. We have used co-immunoprecipitation and mass spectrometry to identify proteins that interact with PABPN1 in mouse skeletal muscles. Among the interacting proteins we identified Matrin 3 (MATR3) as a novel protein interactor of PABPN1. The MATR3 gene is mutated in a form of distal myopathy and amyotrophic lateral sclerosis (ALS). We demonstrate, that like PABPN1, MATR3 is critical for myogenesis. Furthermore, MATR3 controls critical aspects of RNA processing including alternative polyadenylation and intron retention. We provide evidence that MATR3 also binds and regulates the levels of long non-coding RNA (lncRNA) Neat1 and together with PABPN1 is required for normal paraspeckle function. We demonstrate that PABPN1 and MATR3 are required for paraspeckles, as well as for adenosine to inosine (A to I) RNA editing of Ctn RNA in muscle cells. We provide a functional link between PABPN1 and MATR3 through regulation of a common lncRNA target with downstream impact on paraspeckle morphology and function. We extend our analysis to a mouse model of OPMD and demonstrate altered paraspeckle morphology in the presence of endogenous levels of alanine-expanded PABPN1. In this study, we report protein-binding partners of PABPN1, which could provide insight into novel functions of PABPN1 in skeletal muscle and identify proteins that could be sequestered with alanine-expanded PABPN1 in the nuclear aggregates found in OPMD.
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Novel mouse models of oculopharyngeal muscular dystrophy (OPMD) reveal early onset mitochondrial defects and suggest loss of PABPN1 may contribute to pathology
Human molecular genetics, 2017Co-Authors: Katherine E. Vest, Dinghai Zheng, Brittany L. Phillips, Ayan Banerjee, Luciano H. Apponi, Eric B. Dammer, Bin Tian, Grace K. PavlathAbstract:Oculopharyngeal muscular dystrophy (OPMD) is a late onset disease caused by polyalanine expansion in the poly(A) binding protein nuclear 1 (PABPN1). Several mouse models have been generated to study OPMD; however, most of these models have employed transgenic overexpression of alanine-expanded PABPN1. These models do not recapitulate the OPMD patient genotype and PABPN1 overexpression could confound molecular phenotypes. We have developed a knock-in mouse model of OPMD (PABPN1+/A17) that contains one alanine-expanded PABPN1 allele under the control of the native promoter and one wild-type PABPN1 allele. This mouse is the closest available genocopy of OPMD patients. We show that PABPN1+/A17 mice have a mild myopathic phenotype in adult and aged animals. We examined early molecular and biochemical phenotypes associated with expressing native levels of A17-PABPN1 and detected shorter poly(A) tails, modest changes in poly(A) signal (PAS) usage, and evidence of mitochondrial damage in these mice. Recent studies have suggested that a loss of PABPN1 function could contribute to muscle pathology in OPMD. To investigate a loss of function model of pathology, we generated a heterozygous PABPN1 knock-out mouse model (PABPN1+/Δ). Like the PABPN1+/A17 mice, PABPN1+/Δ mice have mild histologic defects, shorter poly(A) tails, and evidence of mitochondrial damage. However, the phenotypes detected in PABPN1+/Δ mice only partially overlap with those detected in PABPN1+/A17 mice. These results suggest that loss of PABPN1 function could contribute to but may not completely explain the pathology detected in PABPN1+/A17 mice.
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An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.
Journal of neuromuscular diseases, 2015Co-Authors: Katherine E. Vest, Ayan Banerjee, Luciano H. Apponi, Grace K. Pavlath, Anita H. CorbettAbstract:Background Oculopharyngeal muscular dystrophy (OPMD), a late onset disorder affecting specific skeletal muscles, is caused by a (GCG)n expansion mutation in the gene encoding the mRNA processing protein, polyadenylate binding protein nuclear 1 (PABPN1). The expansion in PABPN1 leads to an increase in a stretch of N-terminal alanine residues in the PABPN1 protein from the normal 10 to 12-18. Given this modest change, detection of mutant protein has not been possible without the use of tagged constructs. Objective We sought to generate a polyclonal antibody that recognizes alanine-expanded but not wild type PABPN1 with the goal of making possible analysis of expression and localization of alanine-expanded PABPN1. Methods We immunized rabbits with a GST-tagged alanine peptide and tested the resulting serum against alanine-expanded PABPN1 expressed in cell culture as well as in animal models of OPMD. Results The resulting α-alanine antibody detected PABPN1 proteins that contained 14 or more alanine residues. Importantly, the α-alanine antibody could be used to detect alanine-expanded PABPN1 in muscles from either a mouse or Drosophila model of OPMD. Conclusions This α-alanine antibody provides a new tool that will allow for more in-depth study of how alanine expansion affects aggregation, localization, and steady-state levels of alanine-expanded PABPN1 levels in vivo, providing new insight into the molecular mechanisms underlying OPMD.
