Polyadenylate Binding Protein Nuclear 1 Research Articles

Regulation of Kv11.1 Isoform Expression by Polyadenylate Binding Protein Nuclear 1.

The Kv11.1 voltage-gated potassium channel, encoded by the KCNH2 gene, conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative polyadenylation to generate two C-terminal Kv11.1 isoforms in the heart. Utilization of a poly(A) signal in exon 15 produces the full-length, functional Kv11.1a isoform, while intron 9 polyadenylation generates the C-terminally truncated, nonfunctional Kv11.1a-USO isoform. The relative expression of Kv11.1a and Kv11.1a-USO isoforms plays an important role in the regulation of Kv11.1 channel function. In this study, we tested the hypothesis that the RNA polyadenylate binding protein nuclear 1 (PABPN1) interacts with a unique 22 nt adenosine stretch adjacent to the intron 9 poly(A) signal and regulates KCNH2 pre-mRNA alternative polyadenylation and the relative expression of Kv11.1a C-terminal isoforms. We showed that PABPN1 inhibited intron 9 poly(A) activity using luciferase reporter assays, tandem poly(A) reporter assays, and RNA pulldown assays. We also showed that PABPN1 increased the relative expression level of the functional Kv11.1a isoform using RNase protection assays, immunoblot analyses, and patch clamp recordings. Our present findings suggest a novel role for the RNA-binding protein PABPN1 in the regulation of functional and nonfunctional Kv11.1 isoform expression.

Proteomic analysis reveals that wildtype and alanine-expanded nuclear poly(A)-binding protein exhibit differential interactions in skeletal muscle

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.

Nuclear poly(A)-binding protein aggregates misplace a pre-mRNA outside of SC35 speckle causing its abnormal splicing.

A short abnormal polyalanine expansion in the polyadenylate-binding protein nuclear-1 (PABPN1) protein causes oculopharyngeal muscular dystrophy (OPMD). Mutated PABPN1 proteins accumulate as insoluble intranuclear aggregates in muscles of OPMD patients. While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice have been established, the molecular mechanisms which trigger pathological defects in OPMD and the role of aggregates remain to be determined. Using exon array, for the first time we have identified several splicing defects in OPMD. In particular, we have demonstrated a defect in the splicing regulation of the muscle-specific Troponin T3 (TNNT3) mutually exclusive exons 16 and 17 in OPMD samples compared to controls. This splicing defect is directly linked to the SC35 (SRSF2) splicing factor and to the presence of nuclear aggregates. As reported here, PABPN1 aggregates are able to trap TNNT3 pre-mRNA, driving it outside nuclear speckles, leading to an altered SC35-mediated splicing. This results in a decreased calcium sensitivity of muscle fibers, which could in turn plays a role in muscle pathology. We thus report a novel mechanism of alternative splicing deregulation that may play a role in various other diseases with nuclear inclusions or foci containing an RNA binding protein.

500. Gene Therapy Rescues Disease Phenotype in the Oculopharyngeal Muscular Dystrophy Mouse Model

Among triplet expansion diseases, oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant, late-onset muscle disorder characterized by progressive eyelid drooping, swallowing difficulties and proximal limb weakness. OPMD is caused by a short trinucleotide repeat expansion in the polyadenylate-binding protein nuclear 1 (PABPN1) gene that results in an N-terminal expanded polyalanine tract. PABPN1 controls several biological processes such as the length of mRNA poly(A) tails, the mRNA export from the nucleus and the alternative poly(A) site usage. OPMD is characterized by nuclear aggregates of expanded PABPN1, fibrosis and muscle atrophy. Here we demonstrate that treating mice affected by OPMD over 4 months with an AAV gene therapy strategy based on DNA-directed RNA interference to silence the endogenous expPABPN1, combined with the re-expression of a healthy sequence-optimized human PABPN1 gene, significantly reduced the amount of nuclear aggregates in affected muscles, decreased the intramuscular fibrosis and reverted the muscle strength to the level of healthy wild-type muscles. Furthermore, although muscle atrophy was not reverted, the expression of a healthy PABPN1 markedly increased the cross sectional area of muscle fibres. These results obtained in a relevant mammalian animal model of OPMD pave the way for the clinical application of a gene therapy approach as a treatment for OPMD patients.

An Antibody to Detect Alanine-Expanded PABPN1: A New Tool to Study Oculopharyngeal Muscular Dystrophy.

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.

A South African family with oculopharyngeal muscular dystrophy: Clinical and molecular genetic characteristics.

Autosomal dominantly inherited oculopharyngeal muscular dystrophy (OPMD) is caused by a trinucleotide repeat expansion in exon 1 of the polyadenylate binding protein nuclear 1 (PABPN1) gene on chromosome 14q. A large family with OPMD was recently identified in Pretoria, South Africa (SA). Molecular studies revealed a (GCG)11(GCA)3GCG or (GCN)15 mutant allele. The (GCN)15 mutation detected in this family has been described previously in families from Uruguay and Mexico as a founder effect. To our knowledge, this is the first report of an SA Afrikaner family with molecularly confirmed OPMD. The proband, a 64-year-old woman, presented to the neurology outpatient department at Steve Biko Academic Hospital, Pretoria. A sibship of 18 individuals was identified, of whom eight had OPMD. Four patients were interviewed and examined clinically, and electromyographic studies were performed. Molecular analysis of the PABPN1 gene was performed by polymerase chain reaction amplification and direct sequencing of exon 1 in three of the patients. Patients presented with ptosis, external ophthalmoplegia, dysphagia, dysarthria and mild proximal weakness. High foot arches and absent ankle reflexes raised the possibility of peripheral neuropathy, but electromyography showed only mildly low sensory amplitudes, and myopathic units in two patients.

Oculopharyngeal muscular dystrophy – An under-diagnosed disease in China? Report a China-born Chinese with PABPN1 mutation and epidemiology review of the literature

Most reports about oculopharyngeal muscular dystrophy (OPMD) have been contributed by occidental countries, and most of the victims of this disease are racially white. In contrast, this disorder is rarely seen in Asians and has only one African report. Consequently, OPMD has been regarded as a disease of the Western world. The purpose of this paper is to challenge the accuracy of this concept. In a Chinese immigrant family, 3 patients manifesting signs related to OPMD were examined. Electromyography, nerve conduction studies, muscle biopsy and genetic analysis were performed on the proband. All the 322 papers about OPMD were reviewed and their country of origin was labeled to perceive the approximate prevalence of OPMD. Countries were categorized into groups according to the continents to which they belonged. The proband's muscle histopathology showed small angulated fiber with rimmed vacuoles, ultrastructural pathology exposed filamentous intranuclear inclusions, and genetic analysis of the polyadenylate binding protein nuclear 1(PABPN1) gene revealed 13 GCG trinucleotide repeats in one allele (GCG)13 while being normal in the other. The survey of the country of origin of OPMD reports showed that 80% of these papers were contributed by occidental countries and that the number of publications of OPMD among countries of Americas and Asia were unequal, when compared to those of European countries, which were fairly proportioned. An epidemiologic review of the literature is presented and the prevalence of OPMD is discussed. This is a China-born Chinese patient with both morphologically and genetically proven of OPMD. The very low OPMD report rate in developing countries of East Asia is due to the unfamiliarity of medical workers to OPMD and the unavailability of medical supplies to confirm the diagnosis. In addition, the present and previous reports provide clear evidence that OPMD in these areas is underdiagnosed.

Structural basis for a PABPN1 aggregation-preventing antibody fragment in OPMD

Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 α-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5.

Study of a Taiwanese family with oculopharyngeal muscular dystrophy

Background Oculopharyngeal muscular dystrophy (OPMD) is a late onset autosomal dominant muscle disorder. OPMD is caused by a short trinucleotide repeat expansion encoding an expanded polyalanine tract in the polyadenylate binding-protein nuclear 1 ( PABPN1) gene. We identified and characterized a PABPN1 mutation in a Taiwanese family with OPMD. Methods The phenotypic and genotypic characteristics of all subjects were evaluated in a Taiwanese OPMD family. Genetic alterations in the PABPN1 gene were identified using PCR and DNA sequencing. Results Ten subjects with OPMD (6 symptomatic and 4 asymptomatic) within the Taiwanese family carried a novel mutation in the PABPN1 gene. The normal (GCG) 6(GCA) 3GCG sequence was replaced by (GCG) 6(GCA)(GCG) 4(GCA) 3GCG due to an insertion of (GCG) 4GCA into the normal allele in the Taiwanese OPMD subjects. Conclusions In contrast to a single GCG expansion in most of OPMD patients in the literature, an insertion of (GCG) 4GCA in the PABPN1 gene was found in the Taiwanese OPMD subjects. The identification of this mutation appears to support the molecular mechanism of unequal cross-over of two PABPN1 alleles.

Identification of a novel mutation in a Korean patient with oculopharyngeal muscular dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disorder characterized by progressive dysphagia and bilateral ptosis. Mutations in the polyadenylate binding protein nuclear 1 ( PABPN1) gene have been found to cause OPMD. The typical mutation is a stable trinucleotide repeat expansion in the first exon of the PABPN1 gene, in which (GCG) 6 is the normal repeat length. We investigated a Korean patient with OPMD and identified a novel mutation: a heterozygous insertion of a 9-bp sequence [(GCG)(GCA)(GCA); c.27_28insGCGGCAGCA] instead of the (GCG) repeat expansion, resulting in an in-frame insertion of three alanines (p.A10insAAA). To the best of our knowledge, this is the first report of a genetically confirmed case of OPMD in Korea.

A de Novo PABPN1 Germline Mutation in a Patient with Oculopharyngeal Muscular Dystrophy

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset autosomal dominantly inherited disorder characterized by dysphagia, ptosis, and proximal limb weakness and is caused by germline mutations (triplet repeat expansions) in the polyadenylate binding protein nuclear 1 (PABPN1) gene. To describe a 70-year-old female patient with OPMD on the clinical and molecular genetic level and to develop a rapid and efficient molecular genetic screening method to study large patient groups. Detailed family history and clinical assessment of the OPMD patient were followed by mutation analysis of the PABPN1 gene by direct DNA sequencing and by our newly developed method, fluorescent PABPN1 polymerase chain reaction (PCR) product (flPPP) method. A cohort of 50 healthy Swiss probands was screened using the flPPP to assess the frequency of the (GCG)7 allele in the Swiss population. Cricopharyngeal myotomy was performed as treatment for dysphagia. A heterozygous (GCG)9 triplet repeat expansion in PABPN1 was identified. Since the family history proved to be negative, the mutation is likely to have occurred de novo. The frequency of the (GCG)7 allele among healthy Swiss controls amounted to 1%. The flPPP method showed a sensitivity and specificity of 100%. Two years after cricopharyngeal myotomy, the patient is still relieved of dysphagia. An otolaryngologist should include OPMD in the differential diagnosis of a patient presenting with dysphagia, as this symptom can be the first sign of the disease and family history can be negative. Molecular genetic testing represents a highly accurate and rapid way to confirm the clinical diagnosis of OPMD. Cricopharyngeal myotomy relieves the patient of dysphagia in the majority of cases.

Oculopharyngeal muscular dystrophy with PABPN1 mutation in a Chinese Malaysian woman

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder of middle age presenting as progressive dysphagia and eyelid ptosis, due to short expansions of the GCG trinucleotide repeat (from GCG 6 to GCG 8–13) in the polyadenylate binding-protein nuclear 1 (PABPN1) gene. OPMD is rarely seen in Asians and morphologically and/or genetically confirmed cases have been reported in Japanese kindreds only. We report a 64 year old Chinese–Malaysian woman who presented with progressive dysphagia and bilateral ptosis for about 6 years. Her mother and elder brother (both deceased) were believed to be affected. Muscle histopathology revealed angulated fibres with rimmed vacuoles. Genetic analysis showed repeat expansion in one allele to (GCG) 9 while normal in the other (GCG) 6. This is the first non-Japanese Asian family with genetically confirmed OPMD.

Continuous remodeling of adult extraocular muscles as an explanation for selective craniofacial vulnerability in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular mystrophy (OPMD) is an inherited disorder caused by mutations of the polyadenylate binding protein nuclear 1 (PABPN1) gene. While a pathogenic hypothesis has been formulated that links the genetic and molecular abnormalities to cellular abnormalities, there is no proven explanation for the targeting of the craniofacial muscles. We propose a hypothesis that bridges this gap. It is based on the phenomenon of continuous remodeling of normal adult extraocular muscles (EOMs). Unlike the EOMs, the myonuclei of other skeletal muscles are postmitotic in the adult unless the muscles are injured. Continuous myofiber remodeling most likely requires upregulation of genes involved in cell cycling, and in protein degradation and synthesis. PABPN1 is a nuclear protein that performs the essential function of controlling polyadenylation of mRNA and the fidelity of protein synthesis. In OPMD, the ongoing production of mutant PABPN1 in muscles undergoing continuous remodeling could result in a failure of accurate production of mRNA required for the maintenance of the myocytes. Over many years, this would lead to cumulative myonuclear loss and finally to myofiber loss. This hypothesis offers an explanation for the selective involvement of extraocular muscles affected in OPMD and the onset of symptoms in adulthood.