RESUMEN
DNA damage responses have been well characterized with regard to their cell-autonomous checkpoint functions leading to cell cycle arrest, senescence and apoptosis. In contrast, systemic responses to tissue-specific genome instability remain poorly understood. In adult Caenorhabditis elegans worms germ cells undergo mitotic and meiotic cell divisions, whereas somatic tissues are entirely post-mitotic. Consequently, DNA damage checkpoints function specifically in the germ line, whereas somatic tissues in adult C. elegans are highly radio-resistant. Some DNA repair systems such as global-genome nucleotide excision repair (GG-NER) remove lesions specifically in germ cells. Here we investigated how genome instability in germ cells affects somatic tissues in C. elegans. We show that exogenous and endogenous DNA damage in germ cells evokes elevated resistance to heat and oxidative stress. The somatic stress resistance is mediated by the ERK MAP kinase MPK-1 in germ cells that triggers the induction of putative secreted peptides associated with innate immunity. The innate immune response leads to activation of the ubiquitin-proteasome system (UPS) in somatic tissues, which confers enhanced proteostasis and systemic stress resistance. We propose that elevated systemic stress resistance promotes endurance of somatic tissues to allow delay of progeny production when germ cells are genomically compromised.
Asunto(s)
Adaptación Fisiológica/fisiología , Caenorhabditis elegans/fisiología , Daño del ADN , Células Germinativas/inmunología , Células Germinativas/metabolismo , Inmunidad Innata , Estrés Fisiológico/inmunología , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/inmunología , Proteínas de Caenorhabditis elegans/metabolismo , Daño del ADN/genética , Activación Enzimática , Inestabilidad Genómica/genética , Células Germinativas/enzimología , Calor , Inmunidad Innata/genética , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Ubiquitina/metabolismoRESUMEN
Ubiquitination plays a crucial role in neurodevelopment as exemplified by Angelman syndrome, which is caused by genetic alterations of the ubiquitin ligase-encoding UBE3A gene. Although the function of UBE3A has been widely studied, little is known about its paralog UBE3B. By using exome and capillary sequencing, we here identify biallelic UBE3B mutations in four patients from three unrelated families presenting an autosomal-recessive blepharophimosis-ptosis-intellectual-disability syndrome characterized by developmental delay, growth retardation with a small head circumference, facial dysmorphisms, and low cholesterol levels. UBE3B encodes an uncharacterized E3 ubiquitin ligase. The identified UBE3B variants include one frameshift and two splice-site mutations as well as a missense substitution affecting the highly conserved HECT domain. Disruption of mouse Ube3b leads to reduced viability and recapitulates key aspects of the human disorder, such as reduced weight and brain size and a downregulation of cholesterol synthesis. We establish that the probable Caenorhabditis elegans ortholog of UBE3B, oxi-1, functions in the ubiquitin/proteasome system in vivo and is especially required under oxidative stress conditions. Our data reveal the pleiotropic effects of UBE3B deficiency and reinforce the physiological importance of ubiquitination in neuronal development and function in mammals.
Asunto(s)
Blefarofimosis/genética , Blefaroptosis/genética , Discapacidad Intelectual/genética , Ubiquitina-Proteína Ligasas/genética , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Blefarofimosis/diagnóstico , Blefaroptosis/diagnóstico , Encéfalo/patología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Sistema Nervioso Central , Niño , Preescolar , Exoma , Facies , Femenino , Genotipo , Humanos , Lactante , Discapacidad Intelectual/diagnóstico , Imagen por Resonancia Magnética , Masculino , Ratones , Ratones Noqueados , Mutación , Estrés Oxidativo , Síndrome , Ubiquitina-Proteína Ligasas/deficienciaRESUMEN
Sirtuins, a family of histone deacetylases, have a fiercely debated role in regulating lifespan. In contrast with recent observations, here we find that overexpression of sir-2.1, the ortholog of mammalian SirT1, does extend Caenorhabditis elegans lifespan. Sirtuins mandatorily convert NAD(+) into nicotinamide (NAM). We here find that NAM and its metabolite, 1-methylnicotinamide (MNA), extend C. elegans lifespan, even in the absence of sir-2.1. We identify a previously unknown C. elegans nicotinamide-N-methyltransferase, encoded by a gene now named anmt-1, to generate MNA from NAM. Disruption and overexpression of anmt-1 have opposing effects on lifespan independent of sirtuins, with loss of anmt-1 fully inhibiting sir-2.1-mediated lifespan extension. MNA serves as a substrate for a newly identified aldehyde oxidase, GAD-3, to generate hydrogen peroxide, which acts as a mitohormetic reactive oxygen species signal to promote C. elegans longevity. Taken together, sirtuin-mediated lifespan extension depends on methylation of NAM, providing an unexpected mechanistic role for sirtuins beyond histone deacetylation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Longevidad , Niacinamida/metabolismo , Sirtuinas/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Metilación , Niacinamida/química , Sirtuinas/genéticaRESUMEN
Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Insulina/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Proteoma/metabolismo , Células Receptoras Sensoriales/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina/metabolismoRESUMEN
The nervous system coordinates many aspects of body function such as learning, memory, behaviour and locomotion. Therefore, it must develop and maintain an intricate network of differentiated neuronal cells, which communicate efficiently with each other and with non-neuronal target cells. Unlike most somatic cells, differentiated neurons are post-mitotic and characterized by a highly polarized morphology that determines the flow of information. Among other post-translational modifications, the ubiquitination of specific protein substrates was recently shown to have a crucial role in the regulation of neuronal development and differentiation. Here, we review recent findings that illustrate the mechanisms that mediate the temporal and spatial control of neuronal protein turnover by the ubiquitin-proteasome system (UPS), which is crucial for the development and function of the nervous system.
Asunto(s)
Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Procesamiento Proteico-Postraduccional , Ubiquitina/metabolismo , Animales , Humanos , Sinapsis/metabolismoRESUMEN
Asymmetric distribution of cellular components underlies many biological processes, and the localization of mRNAs within domains of the cytoplasm is one important mechanism of establishing and maintaining cellular asymmetry. mRNA localization often involves assembly of large ribonucleoproteins (RNPs) in the cytoplasm. Using an RNA affinity chromatography approach, we investigated localization RNP formation on the vegetal localization element (VLE) of the mRNA encoding Vg1, a Xenopus TGF-beta family member. We identified 40LoVe, an hnRNP D family protein, as a specific VLE binding protein from Xenopus oocytes. Interaction of 40LoVe with the VLE strictly correlates with the ability of the RNA to localize, and antibodies against 40LoVe inhibit vegetal localization in vivo in oocytes. Our results associate an hnRNP D protein with mRNA localization and have implications for several functions mediated by this important protein family.
Asunto(s)
Glicoproteínas/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Oogénesis/fisiología , ARN Mensajero/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Proteínas de Xenopus/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía de Afinidad/métodos , Glicoproteínas/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/clasificación , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Ribonucleoproteínas Nucleares Heterogéneas/clasificación , Ribonucleoproteínas Nucleares Heterogéneas/genética , Datos de Secuencia Molecular , Oocitos/citología , Oocitos/metabolismo , Filogenia , Unión Proteica , Alineación de Secuencia , Factor de Crecimiento Transformador beta/genética , Proteínas de Xenopus/clasificación , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMEN
In eukaryotic cells, proteasomes exist primarily as 26S holoenzymes, the most efficient configuration for ubiquitinated protein degradation. Here, we show that acute oxidative stress caused by environmental insults or mitochondrial defects results in rapid disassembly of 26S proteasomes into intact 20S core and 19S regulatory particles. Consequently, polyubiquitinated substrates accumulate, mitochondrial networks fragment, and cellular reactive oxygen species (ROS) levels increase. Oxidation of cysteine residues is sufficient to induce proteasome disassembly, and spontaneous reassembly from existing components is observed both in vivo and in vitro upon reduction. Ubiquitin-dependent substrate turnover also resumes after treatment with antioxidants. Reversible attenuation of 26S proteasome activity induced by acute mitochondrial or oxidative stress may be a short-term response distinct from adaptation to long-term ROS exposure or changes during aging.
Asunto(s)
Mitocondrias/metabolismo , Estrés Oxidativo , Complejo de la Endopetidasa Proteasomal/metabolismo , Multimerización de Proteína , Animales , Antioxidantes/farmacología , Caenorhabditis elegans/metabolismo , Línea Celular , Cricetinae , Cisteína/metabolismo , Mitocondrias/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina/metabolismoRESUMEN
Mitochondria maintain cellular homeostasis by coordinating ATP synthesis with metabolic activity, redox signaling, and apoptosis. Excessive levels of mitochondria-derived reactive oxygen species (ROS) promote mitochondrial dysfunction, triggering numerous metabolic disorders. However, the molecular basis for the harmful effects of excessive ROS formation is largely unknown. Here, we identify a link between mitochondrial stress and ubiquitin-dependent proteolysis, which supports cellular surveillance both in Caenorhabditis elegans and humans. Worms defective in respiration with elevated ROS levels are limited in turnover of a GFP-based substrate protein, demonstrating that mitochondrial stress affects the ubiquitin/proteasome system (UPS). Intriguingly, we observed similar proteolytic defects for disease-causing IVD and COX1 mutations associated with mitochondrial failure in humans. Together, these results identify a conserved link between mitochondrial metabolism and ubiquitin-dependent proteostasis. Reduced UPS activity during pathological conditions might potentiate disease progression and thus provides a valuable target for therapeutic intervention.
Asunto(s)
Enfermedades Mitocondriales/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ubiquitina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans , Línea Celular , Ciclooxigenasa 1/genética , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes , Humanos , Immunoblotting , Enfermedades Mitocondriales/fisiopatología , Mutagénesis , Compuestos Orgánicos , Fosforilación Oxidativa , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Intraneuronal deposition of aggregated proteins in tauopathies, Parkinson disease, or familial encephalopathy with neuroserpin inclusion bodies (FENIB) leads to impaired protein homeostasis (proteostasis). FENIB represents a conformational dementia, caused by intraneuronal polymerization of mutant variants of the serine protease inhibitor neuroserpin. In contrast to the aggregation process, the kinetic relationship between neuronal proteostasis and aggregation are poorly understood. To address aggregate formation dynamics, we studied FENIB in Caenorhabditis elegans and mice. Point mutations causing FENIB also result in aggregation of the neuroserpin homolog SRP-2 most likely within the ER lumen in worms, recapitulating morphological and biochemical features of the human disease. Intriguingly, we identified conserved protein quality control pathways to modulate protein aggregation both in worms and mice. Specifically, downregulation of the unfolded protein response (UPR) pathways in the worm favors mutant SRP-2 accumulation, while mice overexpressing a polymerizing mutant of neuroserpin undergo transient induction of the UPR in young but not in aged mice. Thus, we find that perturbations of proteostasis through impairment of the heat shock response or altered UPR signaling enhance neuroserpin accumulation in vivo. Moreover, accumulation of neuroserpin polymers in mice is associated with an age-related induction of the UPR suggesting a novel interaction between aging and ER overload. These data suggest that targets aimed at increasing UPR capacity in neurons are valuable tools for therapeutic intervention.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Estrés del Retículo Endoplásmico , Retículo Endoplásmico/metabolismo , Cuerpos de Inclusión/metabolismo , Neuropéptidos/metabolismo , Serpinas/genética , Serpinas/metabolismo , Factores de Edad , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/metabolismo , Respuesta al Choque Térmico , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Neuronas/metabolismo , Neuronas/patología , Neuropéptidos/química , Neuropéptidos/genética , Mutación Puntual , Polimerizacion , Conformación Proteica , Pliegue de Proteína , Serpinas/química , Transducción de Señal , Respuesta de Proteína Desplegada , NeuroserpinaRESUMEN
The maintenance of proteostasis is a fundamental process that encompasses refolding and degradation of unfolded and damaged proteins to enable organismal development (1). In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis by regulating protein turnover. During the past decade, detailed mechanistic insight about the UPS was revealed from extensive studies in mono-cellular systems, such as yeast or tissue culture cells. However, a further challenge is to decipher how ubiquitin-dependent degradation pathways promote cellular differentiation and development of multicellular organisms. In this chapter, we describe an in vivo assay to study protein turnover during development and in differentiated tissues in response to intrinsic and environmental challenges in the multicellular organism Caenorhabditis elegans. This assay is particularly suitable to perform large-scale genetic screens for the identification of novel proteolysis factors and pathways important for developmental processes and opens new avenues for future investigation of tissue- or development-specific proteostasis networks.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteolisis , Animales , Degradación Asociada con el Retículo Endoplásmico , Redes y Vías Metabólicas , Ubiquitina , Enzimas Activadoras de Ubiquitina/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
In eukaryotic cells, the ubiquitin/proteasome system (UPS) is a key determinant of proteostasis as it regulates the turnover of damaged proteins. However, it is still unclear how the UPS integrates intrinsic and environmental challenges to promote organismal development and survival. Here, we set up an in vivo degradation assay to facilitate the genetic identification of ubiquitin-dependent proteolysis pathways in the multicellular organism Caenorhabditis elegans. Using this assay, we found that mild induction of protein-folding stress, which is nontoxic for wild-type worms, strongly reduces ubiquitin-dependent protein turnover. Ubiquitin-mediated degradation is also reduced by metabolic stress, which correlates with life-span extension. Unlike other stress conditions, however, acute heat stress results in enhanced rather than reduced proteolysis. Intriguingly, our study provides the first evidence for the existence of tissue-specific degradation requirements because loss of key regulators of the UPS, such as proteasomal subunits, causes accumulation of the model substrate, depending on the tissue type. Thus, here we establish a screenable degradation assay that allows diverse genetic screening approaches for the identification of novel cell-type-specific proteostasis networks important for developmental processes, stress response, and aging, thereby substantially extending the work on recently described mechanistic UPS reporter studies.
Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitina/metabolismo , Envejecimiento , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Redes Reguladoras de Genes , Genes de Helminto , Genoma de los Helmintos , Immunoblotting/métodos , Mutación , Complejo de la Endopetidasa Proteasomal/genética , Pliegue de Proteína , Transducción de Señal , Estrés Fisiológico , Ubiquitina/genéticaRESUMEN
Protein ubiquitylation is a key post-translational control mechanism contributing to different physiological processes, such as signal transduction and ageing. The size and linkage of a ubiquitin chain, which determines whether a substrate is efficiently targeted for proteasomal degradation, is determined by the interplay between ubiquitylation and deubiquitylation. A conserved factor that orchestrates distinct substrate-processing co-regulators in diverse species is the ubiquitin-selective chaperone CDC-48 (also known as p97). Several deubiquitylation enzymes (DUBs) have been shown to interact with CDC-48/p97, but the mechanistic and physiological relevance of these interactions remained elusive. Here we report a synergistic cooperation between CDC-48 and ATX-3 (the Caenorhabditis elegans orthologue of ataxin-3) in ubiquitin-mediated proteolysis and ageing regulation. Surprisingly, worms deficient for both cdc-48.1 and atx-3 demonstrated extended lifespan by up to 50%, mediated through the insulin-insulin-like growth factor 1 (IGF-1) signalling pathway. As lifespan extension specifically depends on the deubiquitylation activity of ATX-3, our findings identify a mechanistic link between protein degradation and longevity through editing of the ubiquitylation status of substrates involved in insulin-IGF-1 signalling.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Regulación de la Expresión Génica , Enfermedad de Machado-Joseph/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Ataxina-3 , Caenorhabditis elegans , Proteínas de Ciclo Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Insulina/metabolismo , Longevidad , Modelos Biológicos , Mutación , Transducción de Señal , Factores de Tiempo , Técnicas del Sistema de Dos Híbridos , Ubiquitina/metabolismo , Proteína que Contiene ValosinaRESUMEN
Coordination between cell fate specification and cell cycle control in multicellular organisms is essential to regulate cell numbers in tissues and organs during development, and its failure may lead to oncogenesis. In mammalian cells, as part of a general cell cycle checkpoint mechanism, the F-box protein beta-transducin repeat-containing protein (beta-TrCP) and the Skp1/Cul1/F-box complex control the periodic cell cycle fluctuations in abundance of the CDC25A and B phosphatases. Here, we find that the Caenorhabditis elegans beta-TrCP orthologue LIN-23 regulates a progressive decline of CDC-25.1 abundance over several embryonic cell cycles and specifies cell number of one tissue, the embryonic intestine. The negative regulation of CDC-25.1 abundance by LIN-23 may be developmentally controlled because CDC-25.1 accumulates over time within the developing germline, where LIN-23 is also present. Concurrent with the destabilization of CDC-25.1, LIN-23 displays a spatially dynamic behavior in the embryo, periodically entering a nuclear compartment where CDC-25.1 is abundant.
Asunto(s)
Caenorhabditis elegans/fisiología , Regulación del Desarrollo de la Expresión Génica , Intestinos/citología , Animales , Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linaje de la Célula , Núcleo Celular/metabolismo , Proteínas F-Box/metabolismo , Inmunohistoquímica/métodos , Microscopía Confocal/métodos , Modelos Biológicos , Modelos Genéticos , Fenotipo , Interferencia de ARN , Fosfatasas cdc25/metabolismoRESUMEN
PHAX (phosphorylated adaptor for RNA export) is the key regulator of U snRNA nuclear export in metazoa. Our previous work revealed that PHAX is phosphorylated in the nucleus and is exported as a component of the U snRNA export complex to the cytoplasm, where it is dephosphorylated (M. Ohno, A. Segref, A. Bachi, M. Wilm, and I. W. Mattaj, Cell 101:187-198, 2000). PHAX phosphorylation is essential for export complex assembly, whereas its dephosphorylation causes export complex disassembly. Thus, PHAX is subject to a compartmentalized phosphorylation/dephosphorylation cycle that contributes to transport directionality. However, neither essential PHAX phosphorylation sites nor the modifying enzymes that contribute to the compartmentalized system have been identified. Here, we identify PHAX phosphorylation sites that are necessary and sufficient for U snRNA export. Mutation of the phosphorylation sites inhibited U snRNA export in a dominant-negative way. We also show, by both biochemical and RNA interference knockdown experiments, that the nuclear kinase and the cytoplasmic phosphatase for PHAX are CK2 kinase and protein phosphatase 2A, respectively. Our results reveal the composition of the compartmentalized phosphorylation/dephosphorylation system that regulates U snRNA export. This finding was surprising in that such a specific system for U snRNA export regulation is composed of two such universal regulators, suggesting that this compartmentalized system is used more broadly for gene expression regulation.
Asunto(s)
Núcleo Celular/metabolismo , ARN Nuclear Pequeño/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Quinasa de la Caseína II/metabolismo , Línea Celular , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Transporte Nucleocitoplasmático/química , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/aislamiento & purificación , Monoéster Fosfórico Hidrolasas/metabolismo , FosforilaciónRESUMEN
The nuclear cap-binding complex (CBC) binds the 7-methyl-G(5')ppp(5')N cap structure at the 5' end of pre-messenger and uracil-rich small nuclear RNAs in the nucleus. It mediates interaction of these capped RNAs with various nuclear machineries involved in RNA maturation and is co-exported with them to the cytoplasm. The structure of human CBC, which comprises the subunits CBP20 and CBP80, has previously been determined in a mildly trypsinated form which can no longer bind the cap. Here, the engineering and crystallization of two variant CBCs with deletions in CBP80 which do not affect function are described. A complex with a small N-terminal deletion in CBP80 was crystallized in space group C2 with one complex per asymmetric unit. The crystals diffract to 2 A resolution and give the first structure of intact but cap-free CBC. An additional internal deletion in CBP80 of a prominent solvent-exposed coiled coil gives rise to a more compact complex. This was co-crystallized with the cap analogue m(7)GpppG in two different crystal forms which could grow in the same drop. Form 1 belongs to space group P3(1)21 with one complex per asymmetric unit and diffracts to 2.15 A resolution. Form 2 belongs to space group P2(1)2(1)2(1) with two complexes per asymmetric unit and diffracts to 2.3 A resolution. In both forms, strong extra electron density is observed for the cap analogue and for the N- and C-terminal extensions of CBP20 which was absent or disordered in all previous structures.
Asunto(s)
Fosfatos de Dinucleósidos/química , Complejo Proteico Nuclear de Unión a la Caperuza , Fosfoproteínas/química , Ingeniería de Proteínas , Caperuzas de ARN/química , Proteínas de Saccharomyces cerevisiae , Secuencia de Bases , Clonación Molecular , Cristalización , Cartilla de ADN , Humanos , Fosfoproteínas/genética , Fosfoproteínas/aislamiento & purificaciónRESUMEN
Different classes of RNA are exported from the nucleus by distinct factors. We demonstrate that U1 snRNA is exported like an mRNA on insertion of a pre-mRNA intron or either sense or antisense mRNA exon sequences. mRNA-specific factors are recruited onto the spliced or elongated U1 RNA whereas U snRNA-specific factors are not, suggesting that an unstructured region of sufficient length in an RNA acts as a dominant determinant of mRNA identity. After export, spliced U1 RNA undergoes cytoplasmic maturation but is not reimported into the nucleus. These data provide insight into mechanisms for discrimination of different classes of nuclear RNA and demonstrate that two RNAs of identical sequence can have distinct cytoplasmic fates depending on their mode of export.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Núcleo Celular/metabolismo , Proteínas de Transporte Nucleocitoplasmático , Empalme del ARN/genética , ARN Mensajero/metabolismo , ARN Nuclear Pequeño/metabolismo , Animales , Exones/genética , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Intrones/genética , Conformación de Ácido Nucleico , Oocitos/fisiología , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , ARN de Transferencia/metabolismo , Proteínas de Unión al ARN , Empalmosomas/metabolismo , Proteínas de Xenopus , Xenopus laevisRESUMEN
The heterodimeric nuclear cap-binding complex (CBC) binds to the 5' cap structure of RNAs in the nucleus and plays a central role in their diverse maturation steps. We describe the crystal structure at 2.1 A resolution of human CBC bound to an m(7)GpppG cap analogue. Comparison with the structure of uncomplexed CBC shows that cap binding induces co-operative folding around the dinucleotide of some 50 residues from the N- and C-terminal extensions to the central RNP domain of the small subunit CBP20. The cap-bound conformation of CBP20 is stabilized by an intricate network of interactions both to the ligand and within the subunit, as well as new interactions of the CBP20 N-terminal tail with the large subunit CBP80. Although the structure is very different from that of other known cap-binding proteins, such as the cytoplasmic cap-binding protein eIF4E, specificity for the methylated guanosine again is achieved by sandwiching the base between two aromatic residues, in this case two conserved tyrosines. Implications for the transfer of capped mRNAs to eIF4E, required for translation initiation, are discussed.