RESUMEN
Mitochondrial encephalomyopathies (MEs) result from mutations in mitochondrial genes critical to oxidative phosphorylation. Severe and untreatable ME results from mutations affecting each endogenous mitochondrial encoded gene, including all 13 established protein coding genes. Effective techniques to manipulate mitochondrial genome are limited and targeted mitochondrial protein expression is currently unavailable. Here we report the development of a mitochondrial-targeted RNA expression (mtTRES) vector capable of protein expression within mitochondria (mtTRESPro). We demonstrate that mtTRESPro expressed RNAs are targeted to mitochondria and are capable of being translated using EGFP encoded constructs in vivo. We additionally test mtTRESPro constructs encoding wild type ATP6 for their ability to rescue an established ATP61Drosophila model of ME. Genetic rescue is examined including tests with co-expression of mitochondrial targeted translational inhibitors TLI-NCL::ATP6 RNAs that function to reduce expression of the endogenous mutant protein. The data demonstrate allotopic RNA expression of mitochondrial targeted wild type ATP6 coding RNAs are sufficient to partially rescue a severe and established animal model of ME but only when combined with a method to inhibit mutant protein expression, which likely competes for incorporation into complex V.
Asunto(s)
Enfermedades Mitocondriales/genética , Proteínas Mitocondriales/genética , Sistemas de Lectura Abierta/fisiología , ARN Mitocondrial/genética , Animales , Animales Modificados Genéticamente , Células Cultivadas , Drosophila , Células HeLa , Humanos , Enfermedades Mitocondriales/metabolismo , Proteínas Mitocondriales/metabolismo , ARN Mitocondrial/metabolismoRESUMEN
Triosephosphate isomerase (TPI) is a glycolytic enzyme that converts dihydroxyacetone phosphate (DHAP) into glyceraldehyde 3-phosphate (GAP). Glycolytic enzyme dysfunction leads to metabolic diseases collectively known as glycolytic enzymopathies. Of these enzymopathies, TPI deficiency is unique in the severity of neurological symptoms. The Drosophila sugarkill mutant closely models TPI deficiency and encodes a protein prematurely degraded by the proteasome. This led us to question whether enzyme catalytic activity was crucial to the pathogenesis of TPI sugarkill neurological phenotypes. To study TPI deficiency in vivo we developed a genomic engineering system for the TPI locus that enables the efficient generation of novel TPI genetic variants. Using this system we demonstrate that TPI sugarkill can be genetically complemented by TPI encoding a catalytically inactive enzyme. Furthermore, our results demonstrate a non-metabolic function for TPI, the loss of which contributes significantly to the neurological dysfunction in this animal model.
Asunto(s)
Anemia Hemolítica Congénita no Esferocítica/enzimología , Errores Innatos del Metabolismo de los Carbohidratos/enzimología , Drosophila melanogaster/fisiología , Longevidad , Parálisis/enzimología , Triosa-Fosfato Isomerasa/deficiencia , Triosa-Fosfato Isomerasa/metabolismo , Anemia Hemolítica Congénita no Esferocítica/genética , Animales , Errores Innatos del Metabolismo de los Carbohidratos/genética , Catálisis , Dihidroxiacetona Fosfato/metabolismo , Modelos Animales de Enfermedad , Drosophila melanogaster/enzimología , Femenino , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Ingeniería Genética , Gliceraldehído 3-Fosfato/metabolismo , Glucólisis/genética , Calor/efectos adversos , Masculino , Mutación/genética , Parálisis/genética , Estrés Fisiológico/genética , Transgenes/genética , Triosa-Fosfato Isomerasa/genéticaRESUMEN
Endogenous mitochondrial genes encode critical oxidative phosphorylation components and their mutation results in a set of disorders known collectively as mitochondrial encephalomyopathies. There is intensive interest in modulating mitochondrial function as organelle dysfunction has been associated with numerous disease states. Proteins encoded by the mitochondrial genome cannot be genetically manipulated by current techniques. Here we report the development of a mitochondrial-targeted RNA expression system (mtTRES) utilizing distinct non-coding leader sequences (NCLs) and enabling in vivo expression of small mitochondrial-targeted RNAs. mtTRES expressing small chimeric antisense RNAs was used as translational inhibitors (TLIs) to target endogenous mitochondrial protein expression in vivo. By utilizing chimeric antisense RNA we successfully modulate expression of two mitochondrially-encoded proteins, ATP6 and COXII, and demonstrate the utility of this system in vivo and in human cells. This technique has important and obvious research and clinical implications.
Asunto(s)
Proteínas Mitocondriales/metabolismo , Biosíntesis de Proteínas , ARN sin Sentido/genética , ARN Pequeño no Traducido/genética , Envejecimiento/metabolismo , Animales , Animales Modificados Genéticamente , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnicas de Silenciamiento del Gen , Vectores Genéticos , Células HeLa , Humanos , Mitocondrias/metabolismo , Encefalomiopatías Mitocondriales/metabolismo , Proteínas Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Estabilidad del ARN , ARN sin Sentido/metabolismo , ARN Pequeño no Traducido/metabolismoRESUMEN
Mitochondrial dysfunction plays an important role in the pathogenesis of neurodegenerative diseases, numerous other disease states and senescence. The ability to monitor reactive oxygen species (ROS) within tissues and over time in animal model systems is of significant research value. Recently, redox-sensitive fluorescent proteins have been developed. Transgenic flies expressing genetically encoded redox-sensitive GFPs (roGFPs) targeted to the mitochondria function as a useful in vivo assay of mitochondrial dysfunction and ROS. We have generated transgenic flies expressing a mitochondrial-targeted roGFP2, demonstrated its responsiveness to redox changes in cultured cells and in vivo and utilized this protein to discover elevated ROS as a contributor to pathogenesis in a characterized neurodegeneration mutant and in a model of mitochondrial encephalomyopathy. These studies identify the role of ROS in pathogenesis associated with mitochondrial disease and demonstrate the utility of genetically encoded redox sensors in Drosophila.
Asunto(s)
Mitocondrias/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Drosophila melanogaster , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Enfermedades Neurodegenerativas/genética , Neuronas/metabolismo , Oxidación-ReducciónRESUMEN
Triose phosphate isomerase (TPI) deficiency glycolytic enzymopathy is a progressive neurodegenerative condition that remains poorly understood. The disease is caused exclusively by specific missense mutations affecting the TPI protein and clinically features hemolytic anemia, adult-onset neurological impairment, degeneration, and reduced longevity. TPI has a well-characterized role in glycolysis, catalyzing the isomerization of dihydroxyacetone phosphate (DHAP) to glyceraldehyde-3-phosphate (G3P); however, little is known mechanistically about the pathogenesis associated with specific recessive mutations that cause progressive neurodegeneration. Here, we describe key aspects of TPI pathogenesis identified using the TPI(sugarkill) mutation, a Drosophila model of human TPI deficiency. Specifically, we demonstrate that the mutant protein is expressed, capable of forming a homodimer, and is functional. However, the mutant protein is degraded by the 20S proteasome core leading to loss-of-function pathogenesis.
Asunto(s)
Drosophila/enzimología , Drosophila/genética , Genes de Insecto , Triosa-Fosfato Isomerasa/genética , Triosa-Fosfato Isomerasa/metabolismo , Animales , Animales Modificados Genéticamente , Dimerización , Estabilidad de Enzimas , Expresión Génica , Genes Recesivos , Humanos , Longevidad/genética , Longevidad/fisiología , Modelos Genéticos , Actividad Motora/genética , Actividad Motora/fisiología , Mutación Missense , Complejo de la Endopetidasa Proteasomal/metabolismo , Estructura Cuaternaria de Proteína , Triosa-Fosfato Isomerasa/química , Triosa-Fosfato Isomerasa/deficienciaRESUMEN
Mitochondrial encephalomyopathies are common and devastating multisystem genetic disorders characterized by neuromuscular dysfunction and tissue degeneration. Point mutations in the human mitochondrial ATP6 gene are known to cause several related mitochondrial disorders: NARP (neuropathy, ataxia, and retinitis pigmentosa), MILS (maternally inherited Leigh's syndrome), and FBSN (familial bilateral striatal necrosis). We identified a pathogenic mutation in the Drosophila mitochondrial ATP6 gene that causes progressive, adult-onset neuromuscular dysfunction and myodegeneration. Our results demonstrate ultrastructural defects in the mitochondrial innermembrane, neural dysfunction, and a marked reduction in mitochondrial ATP synthase activity associated with this mutation. This Drosophila mutant recapitulates key features of the human neuromuscular disorders enabling detailed in vivo studies of these enigmatic diseases.
Asunto(s)
ADN Mitocondrial/genética , Proteínas de Drosophila/genética , Encefalomiopatías Mitocondriales/enzimología , Encefalomiopatías Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/genética , Animales , Drosophila melanogaster/enzimología , Drosophila melanogaster/genética , Drosophila melanogaster/ultraestructura , Femenino , Masculino , Encefalomiopatías Mitocondriales/patología , Mutación PuntualRESUMEN
Heritable mutations, known as inborn errors of metabolism, cause numerous devastating human diseases, typically as a result of a deficiency in essential metabolic products or the accumulation of toxic intermediates. We have isolated a missense mutation in the Drosophila sugarkill (sgk) gene that causes phenotypes analogous to symptoms of triosephosphate isomerase (TPI) deficiency, a human familial disease, characterized by anaerobic metabolic dysfunction resulting from pathological missense mutations affecting the encoded TPI protein. In Drosophila, the sgk gene encodes the glycolytic enzyme TPI. Our analysis of sgk mutants revealed TPI impairment associated with reduced longevity, progressive locomotor deficiency, and neural degeneration. Biochemical studies demonstrate that mutation of this glycolytic enzyme gene does not result in a bioenergetic deficit, suggesting an alternate cause of enzymopathy associated with TPI impairment.
Asunto(s)
Drosophila/genética , Enfermedades Metabólicas/genética , Errores Innatos del Metabolismo , Triosa-Fosfato Isomerasa/deficiencia , Triosa-Fosfato Isomerasa/genética , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Modelos Animales de Enfermedad , Drosophila/enzimología , Regulación Enzimológica de la Expresión Génica/genética , Humanos , Longevidad/genética , Enfermedades Metabólicas/enzimología , Datos de Secuencia Molecular , Mutación Missense , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Treonina/metabolismo , Transgenes , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
SR proteins are essential pre-mRNA splicing factors that have been shown to bind a number of exonic splicing enhancers where they function to stimulate the splicing of adjacent introns. Members of the SR protein family contain one or two N-terminal RNA binding domains, as well as a C-terminal arginine-serine (RS) rich domain. The RS domains mediate protein-protein interactions with other RS domain containing proteins and are essential for many, but not all, SR protein functions. Hybrid proteins containing an RS domain fused to the bacteriophage MS2 coat protein are sufficient to activate enhancer-dependent splicing in HeLa cell nuclear extract when bound to the pre-mRNA. Here we report progress towards determining the protein sequence requirements for RS domain function. We show that the RS domains from non-SR proteins can also function as splicing activation domains when tethered to the pre-mRNA. Truncation experiments with the RS domain of the human SR protein 9G8 identified a 29 amino acid segment, containing 26 arginine or serine residues, that is sufficient to activate splicing when fused to MS2. We also show that synthetic domains composed solely of RS dipeptides are capable of activating splicing, although their potency is proportional to their size.
Asunto(s)
Precursores del ARN/metabolismo , Empalme del ARN , Secuencias Repetitivas de Ácidos Nucleicos/genética , Secuencia de Aminoácidos , Animales , Arginina/genética , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Línea Celular , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteína Nuclear Pequeña U1/genética , Ribonucleoproteína Nuclear Pequeña U1/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Eliminación de Secuencia , Serina/genética , Empalmosomas/genética , Empalmosomas/metabolismo , Factor de Empalme U2AF , SpodopteraRESUMEN
Lethal giant larvae (Lgl) plays essential and conserved functions in regulating both cell polarity and tumorigenesis in Drosophila melanogaster and vertebrates. It is well recognized that plasma membrane (PM) or cell cortex localization is crucial for Lgl function in vivo, but its membrane-targeting mechanisms remain poorly understood. Here, we discovered that hypoxia acutely and reversibly inhibits Lgl PM targeting through a posttranslational mechanism that is independent of the well-characterized atypical protein kinase C (aPKC) or Aurora kinase-mediated phosphorylations. Instead, we identified an evolutionarily conserved polybasic (PB) domain that targets Lgl to the PM via electrostatic binding to membrane phosphatidylinositol phosphates. Such PB domain-mediated PM targeting is inhibited by hypoxia, which reduces inositol phospholipid levels on the PM through adenosine triphosphate depletion. Moreover, Lgl PB domain contains all the identified phosphorylation sites of aPKC and Aurora kinases, providing a molecular mechanism by which phosphorylations neutralize the positive charges on the PB domain to inhibit Lgl PM targeting.
Asunto(s)
Hipoxia de la Célula/fisiología , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Procesamiento Proteico-Postraduccional/genética , Proteínas Supresoras de Tumor/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Aurora Quinasas/metabolismo , Proteínas de Drosophila/genética , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Datos de Secuencia Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Fosforilación , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Alineación de Secuencia , Electricidad Estática , Proteínas Supresoras de Tumor/genéticaRESUMEN
Single-strand conformational polymorphism analysis has been used successfully to identify single nucleotide changes within sequences based on the fact that multidetection enhancement gels will separate molecules based on their conformation rather than their size. We have expanded the utility of this technique to analyze easily the alternative splicing of pre-mRNAs containing multiple mutually exclusive exons of the same size. We have used this technique to study the Caenorhabditis elegans let-2 gene containing two alternative exons and the Drosophilia melanogaster Dscam gene, which contains 12 mutually exclusive exons. The ease and the quantitative nature of this technique should be very useful.
Asunto(s)
Empalme Alternativo/fisiología , Caenorhabditis elegans/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Electroforesis/métodos , Genes de Helminto/fisiología , Proteínas/aislamiento & purificación , Animales , Caenorhabditis elegans/metabolismo , Moléculas de Adhesión Celular , Drosophila melanogaster/metabolismo , Exones/genética , Intrones/genética , Polimorfismo Conformacional Retorcido-Simple , Isoformas de Proteínas , Proteínas/genética , Precursores del ARN/genéticaRESUMEN
RNA interference (RNAi) is a useful tool for degrading targeted messenger RNAs (mRNAs) and thus "knocking down" the abundance of the encoded protein. We have been using RNAi in cultured Drosophila cells to evaluate the effect of "knocking down" numerous mRNA processing factors on the alternative splicing of specific pre-mRNAs. This relatively simple technique has allowed us to identify a number of splicing factors that impact the alternative splicing of particular alternatively spliced exons. This approach can be extended to examine the splicing of nearly any gene.
Asunto(s)
Empalme Alternativo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interferencia de ARN , ARN Mensajero/metabolismo , Animales , Células Cultivadas , Proteínas de Drosophila/genética , Precursores del ARN/metabolismoRESUMEN
Reactive oxygen species (ROS) play essential roles in cell signaling, survival, and homeostasis. Aberrant ROS lead to disease and contribute to the aging process. Numerous enzymes and vigilant antioxidant pathways are required to regulate ROS for normal cellular health. Mitochondria are a major source of ROS, and mechanisms to prevent elevated ROS during oxidative phosphorylation require super oxide dismutase (SOD) activity. SOD2, also known as MnSOD, is targeted to mitochondria and is instrumental in regulating ROS by conversion of superoxides to hydrogen peroxide, which is further broken down into H(2)O and oxygen. Here, we describe the identification of a novel mutation within the mitochondrial SOD2 enzyme in Drosophila that results in adults with an extremely shortened life span, sensitivity to hyperoxia, and neuropathology. Additional studies demonstrate that this novel mutant, SOD2(bewildered), exhibits abnormal brain morphology, suggesting a critical role for this protein in neurodevelopment. We investigated the basis of this neurodevelopmental defect and discovered an increase in aberrant axonal that could underlie the aberrant neurodevelopment and brain morphology defects. This novel allele, SOD2(bewildered), provides a unique opportunity to study the effects of increased mitochondrial ROS on neural development, axonal targeting, and neural cell degeneration in vivo.
RESUMEN
Numerous mitochondrial DNA mutations cause mitochondrial encephalomyopathy: a collection of related diseases for which there exists no effective treatment. Mitochondrial encephalomyopathies are complex multisystem diseases that exhibit a relentless progression of severity, making them both difficult to treat and study. The pathogenic and compensatory metabolic changes that are associated with chronic mitochondrial dysfunction are not well understood. The Drosophila ATP6(1) mutant models human mitochondrial encephalomyopathy and allows the study of metabolic changes and compensation that occur throughout the lifetime of an affected animal. ATP6(1)animals have a nearly complete loss of ATP synthase activity and an acute bioenergetic deficit when they are asymptomatic, but surprisingly we discovered no chronic bioenergetic deficit in these animals during their symptomatic period. Our data demonstrate dynamic metabolic compensatory mechanisms that sustain normal energy availability and activity despite chronic mitochondrial complex V dysfunction resulting from an endogenous mutation in the mitochondrial DNA. ATP6(1)animals compensate for their loss of oxidative phosphorylation through increases in glycolytic flux, ketogenesis and Kreb's cycle activity early during pathogenesis. However, succinate dehydrogenase activity is reduced and mitochondrial supercomplex formation is severely disrupted contributing to the pathogenesis seen in ATP6(1) animals. These studies demonstrate the dynamic nature of metabolic compensatory mechanisms and emphasize the need for time course studies in tractable animal systems to elucidate disease pathogenesis and novel therapeutic avenues.
Asunto(s)
Modelos Animales de Enfermedad , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Enfermedades Mitocondriales/metabolismo , Enfermedades Mitocondriales/fisiopatología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , Conducta Animal , Respiración de la Célula , Ciclo del Ácido Cítrico , Progresión de la Enfermedad , Transporte de Electrón , Metabolismo Energético , Glucólisis , Humanos , Longevidad , Enfermedades Mitocondriales/patología , Modelos Biológicos , Fenotipo , Multimerización de Proteína , Análisis de Supervivencia , Factores de TiempoAsunto(s)
Empalme Alternativo , Proteínas de Drosophila/genética , Exones , ARN Mensajero/metabolismo , Animales , Secuencia de Bases , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Datos de Secuencia Molecular , ARN Mensajero/genética , Homología de Secuencia de Ácido NucleicoRESUMEN
The fruit fly, Drosophila melanogaster, is a powerful model genetic organism that has been used since the turn of the previous century in the study of complex biological problems. In the last decade, numerous researchers have focused their attention on understanding neurodegenerative diseases by utilizing this model system. Numerous Drosophila mutants have been isolated that profoundly affect neural viability and integrity of the nervous system with age. Additionally, many transgenic strains have been developed as models of human disease conditions. We review the existing Drosophila neurodegenerative mutants and transgenic disease models, and discuss the role of the fruit fly in therapeutic development for neurodegenerative diseases.
Asunto(s)
Modelos Animales de Enfermedad , Drosophila/genética , Enfermedades Neurodegenerativas , Animales , Animales Modificados Genéticamente , Drosophila/fisiología , Humanos , Mutación , Enfermedades Neurodegenerativas/tratamiento farmacológico , Enfermedades Neurodegenerativas/genéticaRESUMEN
The goal of functional genomics is to determine the function of each protein encoded by an organism. Typically, this is done by inactivating individual genes and, subsequently, analyzing the phenotype of the modified organisms. In higher eukaryotes, where a tremendous amount of alternative splicing occurs, such approaches are not feasible because they have the potential to simultaneously affect multiple proteins that could have quite distinct and important functions. Thus, it is necessary to develop techniques that inactivate only a subset of proteins synthesized from genes encoding alternatively spliced mRNAs. Here we demonstrate that RNA interference (RNAi) can be used to selectively degrade specific alternatively spliced mRNA isoforms in cultured Drosophila cells. This is achieved by treating the cells with double-stranded RNA corresponding to an alternatively spliced exon. This technique may prove to be a powerful tool to assess the function of proteins synthesized from alternatively spliced mRNAs. In addition, these results have implications regarding the mechanism of RNAi in Drosophila.
Asunto(s)
Empalme Alternativo , Exones , ARN Mensajero/metabolismo , Animales , Drosophila , ARN Mensajero/genéticaRESUMEN
Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing.