RESUMEN
OBJECTIVE: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset inherited neuromuscular disorder, with progressive ptosis and dysphagia as common manifestations. To date, OPMD has rarely been reported among East Asians. The present study summarizes the phenotypic and genotypic features of Chinese patients with OPMD. METHODS: Twenty-one patients with molecularly confirmed OPMD from 9 unrelated families were identified by direct sequencing of the polyadenlyate binding protein nuclear-1 (PABPN1) gene. Immunofluorescence staining of muscle biopsies was conducted to identify the components of protein degradation pathways involved in OPMD. RESULTS: In our cohort, the genetically confirmed OPMD group had a mean age at onset of 50.6 ± 4.2 years (range 45-60 years). Ptosis (42.9%) was the most common initial symptom; patients with ptosis as the first symptom subsequently developed dysphagia within a median time of 5.5 years (range 1-19 years). Evidence of external ophthalmoplegia was found in 38.1% of patients. A total of 33.3% of the patients developed muscle weakness at a median age at onset of 66 years (range 50-70 years), with neck flexor involvement in all patients. Five genotypes were observed in our cohort, including classical (GCG)9-11 repeats in 7 families and non-GCG elongations with additional GCA expansions in 2 families. OPMD muscle biopsies revealed rimmed vacuoles and intranuclear filamentous inclusions. The PABPN1 protein showed substantial accumulation in the nuclei of muscle fiber aggregates and closely colocalized with p62, LC3B and FK2. INTERPRETATION: Our findings indicate wide genetic heterogeneity in OPMD in the Chinese population and demonstrate abnormalities in protein degradation pathways in this disease.
Asunto(s)
Trastornos de Deglución , Distrofia Muscular Oculofaríngea , Humanos , Persona de Mediana Edad , Anciano , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patología , Pueblos del Este de Asia , Genotipo , Proteína II de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/genéticaRESUMEN
BACKGROUND: Polyadenylation plays a key role in producing mature mRNAs in eukaryotes. It is widely believed that the poly(A)-binding proteins (PABs) uniformly bind to poly(A)-tailed mRNAs, regulating their stability and translational efficiency. RESULTS: We observe that the homozygous triple mutant of broadly expressed Arabidopsis thaliana PABs, AtPAB2, AtPAB4, and AtPAB8, is embryonic lethal. To understand the molecular basis, we characterize the RNA-binding landscape of these PABs. The AtPAB-binding efficiency varies over one order of magnitude among genes. To identify the sequences accounting for the variation, we perform poly(A)-seq that directly sequences the full-length poly(A) tails. More than 10% of poly(A) tails contain at least one guanosine (G); among them, the G-content varies from 0.8 to 28%. These guanosines frequently divide poly(A) tails into interspersed A-tracts and therefore cause the variation in the AtPAB-binding efficiency among genes. Ribo-seq and genome-wide RNA stability assays show that AtPAB-binding efficiency of a gene is positively correlated with translational efficiency rather than mRNA stability. Consistently, genes with stronger AtPAB binding exhibit a greater reduction in translational efficiency when AtPAB is depleted. CONCLUSIONS: Our study provides a new mechanism that translational efficiency of a gene can be regulated through the G-content-dependent PAB binding, paving the way for a better understanding of poly(A) tail-associated regulation of gene expression.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Unión a Poli(A)/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/química , ARN Mensajero/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiología , Composición de Base , Genes de Plantas , Guanosina/análisis , Proteína II de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/metabolismo , Proteína II de Unión a Poli(A)/fisiología , Proteínas de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/fisiología , Unión ProteicaRESUMEN
The Hippo tumor-suppressor pathway regulates organ growth, cell proliferation, and stem cell biology. Defects in Hippo signaling and hyperactivation of its downstream effectors-Yorkie (Yki) in Drosophila and YAP/TAZ in mammals-result in progenitor cell expansion and overgrowth of multiple organs and contribute to cancer development. Deciphering the mechanisms that regulate the activity of the Hippo pathway is key to understanding its function and for therapeutic targeting. However, although the Hippo kinase cascade and several other upstream inputs have been identified, the mechanisms that regulate Yki/YAP/TAZ activity are still incompletely understood. To identify new regulators of Yki activity, we screened in Drosophila for suppressors of tissue overgrowth and Yki activation caused by overexpression of atypical protein kinase C (aPKC), a member of the apical cell polarity complex. In this screen, we identified mutations in the heterogeneous nuclear ribonucleoprotein Hrb27C that strongly suppressed the tissue defects induced by ectopic expression of aPKC. Hrb27C was required for aPKC-induced tissue growth and Yki target gene expression but did not affect general gene expression. Genetic and biochemical experiments showed that Hrb27C affects Yki phosphorylation. Other RNA-binding proteins known to interact with Hrb27C for mRNA transport in oocytes were also required for normal Yki activity, although they suppressed Yki output. Based on the known functions of Hrb27C, we conclude that Hrb27C-mediated control of mRNA splicing, localization, or translation is essential for coordinated activity of the Hippo pathway.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Transactivadores/metabolismo , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Proteínas Nucleares/genética , Proteína II de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/metabolismo , Proteínas de Unión al ARN/genética , Transducción de Señal , Transactivadores/genética , Proteínas Señalizadoras YAPRESUMEN
Poly(A)-binding proteins (PABPs) regulate mRNA fate by controlling stability and translation through interactions with both the poly(A) tail and eIF4F complex. Many organisms have several paralogs of PABPs and eIF4F complex components and it is likely that different eIF4F/PABP complex combinations regulate distinct sets of mRNAs. Trypanosomes have five eIF4G paralogs, six of eIF4E and two PABPs, PABP1 and PABP2. Under starvation, polysomes dissociate and the majority of mRNAs, most translation initiation factors and PABP2 reversibly localise to starvation stress granules. To understand this more broadly we identified a protein interaction cohort for both T. brucei PABPs by cryo-mill/affinity purification-mass spectrometry. PABP1 very specifically interacts with the previously identified interactors eIF4E4 and eIF4G3 and few others. In contrast PABP2 is promiscuous, with a larger set of interactors including most translation initiation factors and most prominently eIF4G1, with its two partners TbG1-IP and TbG1-IP2. Only RBP23 was specific to PABP1, whilst 14 RNA-binding proteins were exclusively immunoprecipitated with PABP2. Significantly, PABP1 and associated proteins are largely excluded from starvation stress granules, but PABP2 and most interactors translocate to granules on starvation. We suggest that PABP1 regulates a small subpopulation of mainly small-sized mRNAs, as it interacts with a small and distinct set of proteins unable to enter the dominant pathway into starvation stress granules and localises preferentially to a subfraction of small polysomes. By contrast PABP2 likely regulates bulk mRNA translation, as it interacts with a wide range of proteins, enters stress granules and distributes over the full range of polysomes.
Asunto(s)
Proteína II de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteínas Protozoarias/metabolismo , ARN Mensajero/metabolismo , Trypanosoma brucei brucei/metabolismo , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Proteína I de Unión a Poli(A)/química , Proteína I de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Unión Proteica , Proteómica , Proteínas Protozoarias/química , Proteínas Protozoarias/genética , ARN Mensajero/genética , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genéticaRESUMEN
The chromatoid body (CB) is a specific cloud-like structure in the cytoplasm of haploid spermatids. Recent findings indicate that CB is identified as a male germ cell-specific RNA storage and processing center, but its function has remained elusive for decades. In somatic cells, KH-type splicing regulatory protein (KSRP) is involved in regulating gene expression and maturation of select microRNAs (miRNAs). However, the function of KSRP in spermatogenesis remains unclear. In this study, we showed that KSRP partly localizes in CB, as a component of CB. KSRP interacts with proteins (mouse VASA homolog (MVH), polyadenylate-binding protein 1 (PABP1) and polyadenylate-binding protein 2 (PABP2)), mRNAs (Tnp2 and Odf1) and microRNAs (microRNA-182) in mouse CB. Moreover, KSRP may regulate the integrity of CB via DDX5-miRNA-182 pathway. In addition, we found abnormal expressions of CB component in testes of Ksrp-knockout mice and of patients with hypospermatogenesis. Thus, our results provide mechanistic insight into the role of KSRP in spermatogenesis.
Asunto(s)
Proteínas de Unión al ARN/metabolismo , Espermátides/metabolismo , Espermatogénesis , Transactivadores/metabolismo , Adulto , Animales , Proteínas Argonautas/deficiencia , Proteínas Argonautas/genética , Estudios de Casos y Controles , Células Cultivadas , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Masculino , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Oligospermia/genética , Oligospermia/metabolismo , Proteína I de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/metabolismo , Proteína II de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transducción de Señal , Transactivadores/deficiencia , Transactivadores/genética , Adulto JovenRESUMEN
Cytoplasmic poly(A)-binding proteins (PABPs) link mRNA 3' termini to translation initiation factors, but they also play key roles in mRNA regulation and decay. Reports from mice, zebrafish and Drosophila further involved PABPs in microRNA (miRNA)-mediated silencing, but through seemingly distinct mechanisms. Here, we implicate the two Caenorhabditis elegans PABPs (PAB-1 and PAB-2) in miRNA-mediated silencing, and elucidate their mechanisms of action using concerted genetics, protein interaction analyses, and cell-free assays. We find that C. elegans PABPs are required for miRNA-mediated silencing in embryonic and larval developmental stages, where they act through a multi-faceted mechanism. Depletion of PAB-1 and PAB-2 results in loss of both poly(A)-dependent and -independent translational silencing. PABPs accelerate miRNA-mediated deadenylation, but this contribution can be modulated by 3'UTR sequences. While greater distances with the poly(A) tail exacerbate dependency on PABP for deadenylation, more potent miRNA-binding sites partially suppress this effect. Our results refine the roles of PABPs in miRNA-mediated silencing and support a model wherein they enable miRNA-binding sites by looping the 3'UTR poly(A) tail to the bound miRISC and deadenylase.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Larva/genética , MicroARNs/genética , Poli A/genética , Proteína II de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/genética , Regiones no Traducidas 3' , Adenosina Monofosfato/metabolismo , Animales , Sitios de Unión , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Embrión no Mamífero , Silenciador del Gen , Larva/crecimiento & desarrollo , Larva/metabolismo , MicroARNs/metabolismo , Poli A/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Unión Proteica , Biosíntesis de Proteínas , Complejo Silenciador Inducido por ARN/genética , Complejo Silenciador Inducido por ARN/metabolismoRESUMEN
Most Cryptococccus neoformans genes are interrupted by introns, and alternative splicing occurs very often. In this study, we examined the influence of introns on C. neoformans gene expression. For most tested genes, elimination of introns greatly reduces mRNA accumulation. Strikingly, the number and the position of introns modulate the gene expression level in a cumulative manner. A screen for mutant strains able to express functionally an intronless allele revealed that the nuclear poly(A) binding protein Pab2 modulates intron-dependent regulation of gene expression in C. neoformans. PAB2 deletion partially restored accumulation of intronless mRNA. In addition, our results demonstrated that the essential nucleases Rrp44p and Xrn2p are implicated in the degradation of mRNA transcribed from an intronless allele in C. neoformans. Double mutant constructions and over-expression experiments suggested that Pab2p and Xrn2p could act in the same pathway whereas Rrp44p appears to act independently. Finally, deletion of the RRP6 or the CID14 gene, encoding the nuclear exosome nuclease and the TRAMP complex associated poly(A) polymerase, respectively, has no effect on intronless allele expression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Intrones/genética , Proteína II de Unión a Poli(A)/genética , Estabilidad del ARN/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cryptococcus neoformans/genética , Redes y Vías Metabólicas/genética , Poli A/genética , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The number of paralogs of proteins involved in translation initiation is larger in trypanosomes than in yeasts or many metazoan and includes two poly(A) binding proteins, PABP1 and PABP2, and four eIF4E variants. In many cases, the paralogs are individually essential and are thus unlikely to have redundant functions although, as yet, distinct functions of different isoforms have not been determined. Here, trypanosome PABP1 and PABP2 have been further characterised. PABP1 and PABP2 diverged subsequent to the differentiation of the Kinetoplastae lineage, supporting the existence of specific aspects of translation initiation regulation. PABP1 and PABP2 exhibit major differences in intracellular localization and distribution on polysome fractionation under various conditions that interfere with mRNA metabolism. Most striking are differences in localization to the four known types of inducible RNP granules. Moreover, only PABP2 but not PABP1 can accumulate in the nucleus. Taken together, these observations indicate that PABP1 and PABP2 likely associate with distinct populations of mRNAs. The differences in localization to inducible RNP granules also apply to paralogs of components of the eIF4F complex: eIF4E1 showed similar localization pattern to PABP2, whereas the localisation of eIF4E4 and eIF4G3 resembled that of PABP1. The grouping of translation initiation as either colocalizing with PABP1 or with PABP2 can be used to complement interaction studies to further define the translation initiation complexes in kinetoplastids.
Asunto(s)
Factor 4E Eucariótico de Iniciación/metabolismo , Evolución Molecular , Proteína I de Unión a Poli(A)/metabolismo , Trypanosoma brucei brucei/genética , Núcleo Celular/ultraestructura , Citoplasma/ultraestructura , Humanos , Filogenia , Proteína I de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/metabolismo , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribonucleoproteínas/genética , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Nuclear poly(A)-binding proteins (PABPs) are evolutionarily conserved proteins that play key roles in eukaryotic gene expression. In the fission yeast Schizosaccharomyces pombe, the major nuclear PABP, Pab2, functions in the maturation of small nucleolar RNAs as well as in nuclear RNA decay. Despite knowledge about its nuclear functions, nothing is known about how Pab2 is imported into the nucleus. Here, we show that Pab2 contains a proline-tyrosine nuclear localization signal (PY-NLS) that is necessary and sufficient for its nuclear localization and function. Consistent with the role of karyopherin ß2 (Kapß2)-type receptors in the import of PY-NLS cargoes, we show that the fission yeast ortholog of human Kapß2, Kap104, binds to recombinant Pab2 and is required for Pab2 nuclear localization. The absence of arginine methylation in a basic region N-terminal to the PY-core motif of Pab2 did not affect its nuclear localization. However, in the context of a sub-optimal PY-NLS, we found that Pab2 was more efficiently targeted to the nucleus in the absence of arginine methylation, suggesting that this modification can affect the import kinetics of a PY-NLS cargo. Although a sequence resembling a PY-NLS motif can be found in the human Pab2 ortholog, PABPN1, our results indicate that neither a functional PY-NLS nor Kapß2 activity are required to promote entry of PABPN1 into the nucleus of human cells. Our findings describe the mechanism by which Pab2 is imported into the nucleus, providing the first example of a PY-NLS import system in fission yeast. In addition, this study suggests the existence of alternative or redundant nuclear import pathways for human PABPN1.
Asunto(s)
Núcleo Celular/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , beta Carioferinas/metabolismo , Transporte Activo de Núcleo Celular , Secuencias de Aminoácidos , Arginina/metabolismo , Sitios de Unión , Células HeLa , Humanos , Señales de Localización Nuclear , Proteína I de Unión a Poli(A)/química , Proteína I de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/genética , Prolina/química , Transporte de Proteínas , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Tirosina/química , beta Carioferinas/genéticaRESUMEN
BACKGROUND/PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
Asunto(s)
Distrofia Muscular Oculofaríngea/epidemiología , Proteína II de Unión a Poli(A)/genética , Proteína I de Unión a Poli(A)/genética , Adulto , Anciano , Américas/epidemiología , Asia/epidemiología , China/epidemiología , Electromiografía , Europa (Continente)/epidemiología , Femenino , Técnicas de Genotipaje , Humanos , Masculino , Persona de Mediana Edad , Distrofia Muscular Oculofaríngea/diagnóstico , Distrofia Muscular Oculofaríngea/genética , Conducción Nerviosa/fisiología , Prevalencia , Repeticiones de Trinucleótidos/genéticaRESUMEN
Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.
Asunto(s)
Péptidos/química , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Biocatálisis , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Prolina , Ratas , Especificidad por SustratoRESUMEN
Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.
Asunto(s)
Distrofia Muscular Oculofaríngea/genética , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Deficiencias en la Proteostasis/genética , Alanina/química , Amiloidosis/genética , Amiloidosis/metabolismo , Escherichia coli/genética , Humanos , Distrofia Muscular Oculofaríngea/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Pliegue de Proteína , Estructura Terciaria de Proteína , Deficiencias en la Proteostasis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrofotometría InfrarrojaRESUMEN
Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA.
Asunto(s)
Proteína II de Unión a Poli(A)/metabolismo , Poliadenilación , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína II de Unión a Poli(A)/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
The PABPN1 [nuclear poly(A)-binding protein 1] is ubiquitous, binds to the nascent mRNA transcript and controls the poly(A) tract elongation process in multicellular organisms. Expansion of GCG repeats that encode first 6 of the 10 alanine residues of a polyalanine tract at the N-terminus of wild-type PABPN1 to 12-17 alanine residues causes aggregation of the protein and cell death. Patients with the adult onset autosomal dominant OPMD (oculopharyngeal muscular dystrophy) carry the GCG expansion mutation in their PABPN1 gene. The symptoms of OPMD include drooping eye lids and difficulty swallowing. The severity of symptoms increases with the length of the expansion. We have investigated the mechanism of cell death in HeLa and HEK-293 (human embryonic kidney) cultured cells expressing the mutant PABPN1 with a polyalanine tract containing 17 alanine residues (PABPN1-A17). In cells expressing PABPN1-A17, the abundance of pro-apoptotic proteins, p53, PUMA (p53 up-regulated modulator of apoptosis) and Noxa, are up-regulated. This was associated with the redistribution of p53 to the nucleus and mitochondria. Concomitantly Bax was translocated to the mitochondria, followed by the release of cytochrome c and the cleavage of caspase 3. Furthermore, blocking p53-mediated transcription using pifithrin significantly reduced apoptosis. Our findings suggest a key role of p53-mediated apoptosis in death of cells expressing the polyalanine expansion mutant of PABPN1.
Asunto(s)
Apoptosis , Péptidos/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Benzotiazoles/metabolismo , Caspasa 3/metabolismo , Citocromos c/metabolismo , Células HEK293 , Células HeLa , Humanos , Mitocondrias/metabolismo , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Distrofia Muscular Oculofaríngea/patología , Mutación , Proteína II de Unión a Poli(A)/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Tolueno/análogos & derivados , Tolueno/metabolismo , Regulación hacia Arriba , Proteína X Asociada a bcl-2/metabolismoRESUMEN
INTRODUCTION: Oculopharyngeal muscular dystrophy (OPMD) presents with progressive ptosis, dysphagia and limb girdle weakness, and is caused by expansion of a trinucleotide tandem repeat within the gene encoding poly-(A) binding protein 2. AIM: To review the clinical manifestations of all genetically confirmed patients with OPMD in Scotland identified since 2002, and to estimate the delay between symptom onset and diagnosis. Method Retrospective case note review. RESULTS: The authors identified 17 patients. The commonest first symptom was ptosis at about the age of 60 years. Three to 20 years elapsed from the onset of ptosis to OPMD diagnosis. In 14 (82%) patients, dysphagia had developed by the time of diagnosis, and four (24%) out of these 14 patients with dysphagia had undergone a decade of investigation and treatment for pharyngeal problems. Thirteen patients (77%) also had symptoms of limb girdle muscle weakness. Every patient had a first-degree relative with ptosis. CONCLUSIONS: OPMD could have been diagnosed earlier in every patient in this case series. Greater awareness of OPMD among ophthalmologists, gastroenterologists and otolaryngologists may lead to earlier diagnosis, improved management and avoidance of unnecessary investigations.
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Distrofia Muscular Oculofaríngea/diagnóstico , Anciano , Anciano de 80 o más Años , Blefaroptosis/diagnóstico , Blefaroptosis/genética , Trastornos de Deglución/diagnóstico , Trastornos de Deglución/genética , Diagnóstico Tardío , Femenino , Humanos , Masculino , Persona de Mediana Edad , Debilidad Muscular/diagnóstico , Debilidad Muscular/genética , Distrofia Muscular Oculofaríngea/genética , Proteína II de Unión a Poli(A)/genética , Estudios Retrospectivos , EscociaRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disorder caused by a small expansion of a short polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). It presents with adult onset of progressive eyelid drooping, swallowing difficulties and proximal limb weakness, usually without involvement of central nervous system (CNS). However, cognitive decline with relevant behavioural and psychological symptoms has been recently described in homozygous patients. In this study, we performed for the first time an extensive neuropsychological and neuropsychiatric evaluation on 11 OPMD heterozygote patients. We found that they were less efficient than a matched control sample on several tests, particularly those tapping executive functions. Moreover, the presence of negative correlation between GCN expansion size and some neuropsychological scores raises the issue that CNS involvement might be linked to the genetic defect, being worse in patients with larger expansion. Our results might be consistent with the toxic gain-of-function theory in the pathogenesis of OPMD and hint at a possible direct role of PABPN1 in the CNS also in heterozygote patients.
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Trastornos del Conocimiento/etiología , Función Ejecutiva/fisiología , Heterocigoto , Distrofia Muscular Oculofaríngea/complicaciones , Distrofia Muscular Oculofaríngea/genética , Proteína II de Unión a Poli(A)/genética , Adulto , Anciano , Anciano de 80 o más Años , Estudios de Casos y Controles , Electromiografía , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pruebas Neuropsicológicas , Escalas de Valoración Psiquiátrica , Estadísticas no Paramétricas , Expansión de Repetición de Trinucleótido/genéticaRESUMEN
General discard pathways eliminate unprocessed and irregular pre-mRNAs to control the quality of gene expression. In contrast to such general pre-mRNA decay, we describe here a nuclear pre-mRNA degradation pathway that controls the expression of select intron-containing genes. We show that the fission yeast nuclear poly(A)-binding protein, Pab2, and the nuclear exosome subunit, Rrp6, are the main factors involved in this polyadenylation-dependent pre-mRNA degradation pathway. Transcriptome analysis and intron swapping experiments revealed that inefficient splicing is important to dictate susceptibility to Pab2-dependent pre-mRNA decay. We also show that negative splicing regulation can promote the poor splicing efficiency required for this pre-mRNA decay pathway, and in doing so, we identified a mechanism of cross-regulation between paralogous ribosomal proteins through nuclear pre-mRNA decay. Our findings unveil a layer of regulation in the nucleus in which the turnover of specific pre-mRNAs, besides the turnover of mature mRNAs, is used to control gene expression.
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Exorribonucleasas/genética , Regulación Fúngica de la Expresión Génica , Intrones , Poli A/química , Proteína II de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/metabolismo , Precursores del ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Núcleo Celular/metabolismo , Complejo Multienzimático de Ribonucleasas del Exosoma , Regulación de la Expresión Génica , Modelos Genéticos , Poliadenilación , Empalme del ARN , Ribosomas/metabolismo , Schizosaccharomyces/metabolismo , Factores de TiempoRESUMEN
Increased aggregation of misfolded proteins is associated with aging, and characterizes a number of neurodegenerative disorders caused by homopolymeric amino acid expansion mutations. PABPN1 is an aggregation-prone nuclear protein. Natural aggregation of wild-type (WT) PABPN1 is not known to be disease-associated, but alanine-expanded PABPN1 (expPABPN1) accumulates in insoluble intranuclear inclusions in muscle of patients with oculopharyngeal muscular dystrophy (OPMD). We applied microscopic image quantification to study PABPN1 aggregation process in living cells. We identified transitional pre-inclusion foci and demonstrate that these structures significantly differ between WT- and expPABPN1-expressing cells, while inclusions of these proteins are indistinguishable. In addition to the immobile PABPN1 in inclusions, in the nucleoplasm of expPABPN1 expressing cells we also found a fraction of immobile proteins, representing pre-aggregated species. We found that pre-aggregated and pre-inclusion structures are reverted by a PABPN1 specific affinity binder while inclusion structures are not. Together our results demonstrate that the aggregation process of WT- and expPABPN1 differs in steps preceding inclusion formation, suggesting that pre-aggregated protein species could represent the cytotoxic structures.
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Cuerpos de Inclusión Intranucleares/metabolismo , Proteína II de Unión a Poli(A)/química , Multimerización de Proteína , Línea Celular Tumoral , Supervivencia Celular , Humanos , Cuerpos de Inclusión Intranucleares/genética , Mutación , Proteína II de Unión a Poli(A)/genética , Proteína II de Unión a Poli(A)/metabolismo , Estructura Cuaternaria de Proteína , Factores de TiempoRESUMEN
Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.