RESUMEN
A key aspect of development in all metazoans is remodeling at the cellular level. During the development of gametes, remodeling occurs throughout the germ line. When Caenorhabditis elegans hermaphrodites become depleted of sperm after 4 days of adulthood, significant cellular remodeling occurs within the meiotically-arrested oocytes, including the formation of ribonucleoprotein granules. Since major remodeling of the endoplasmic reticulum (ER) occurs in early embryos, we investigated the extent of ER remodeling in meiotically-arrested oocytes. We found, using a combination of fluorescence reporters and transmission electron microscopy, that the ER in arrested oocytes accumulates in patches and sheets that are enriched at the cortex. Our findings suggest this remodeling is not due to simple displacement by large amounts of yolk that accumulate in arrested oocytes, and instead may be genetically regulated. We further identified the Ddx6 RNA helicase, CGH-1, as a key regulator of ER in the germ line. In cgh-1(tn691) oocytes, we detected cortical ER patches as well as aberrant granules of the RNA-binding proteins, PAB-1, MEX-3, and CGH-1. Taken together, our results suggest the possibility that the spatial organization of RNA binding proteins may regulate the translation of mRNAs associated with the ER that in turn, controls the organization of the ER in the adult germ line.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Retículo Endoplásmico/metabolismo , Oocitos/metabolismo , ARN Nucleotidiltransferasas/metabolismo , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteína I de Unión a Poli(A)/metabolismo , ARN Nucleotidiltransferasas/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Cells respond to environmental stress in multiple ways. In the germ line, heat shock and nutritive stress trigger the assembly of large ribonucleoprotein (RNP) granules via liquid-liquid phase separation (LLPS). The RNP granules are hypothesized to maintain the quality of oocytes during stress. The goal of this study was to investigate the cellular response to glucose in the germ line and determine if it is an osmotic stress response. We found that exposure to 500 mM glucose induces the assembly of RNP granules in the germ line within 1 h. Interestingly, the RNP granules are maintained for up to 3 h; however, they dissociate after longer periods of stress. The RNP granules include processing body and stress granule proteins, suggesting shared functions. Based on several lines of evidence, the germ line response to glucose largely appears to be an osmotic stress response, thus identifying osmotic stress as a trigger of LLPS. Although RNP granules are not maintained beyond 3 h of osmotic stress, the quality of oocytes does not appear to decrease after longer periods of stress, suggesting a secondary adaptation in the germ line. We used an indirect marker of glycerol and observed high levels after 5 and 20 h of glucose exposure. Moreover, in gpdh-1;gpdh-2 germ lines, glycerol levels are reduced concomitant with RNP granules being maintained for an extended period. We speculate that increased glycerol levels may function as a secondary osmoregulatory adaptive response in the germ line, following a primary response of RNP granule assembly.
Asunto(s)
Adaptación Fisiológica , Caenorhabditis elegans/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Glucosa/farmacología , Oocitos/efectos de los fármacos , Ribonucleoproteínas/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Supervivencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/ultraestructura , Trastornos del Desarrollo Sexual/genética , Trastornos del Desarrollo Sexual/metabolismo , Relación Dosis-Respuesta a Droga , Regulación de la Expresión Génica , Genes Reporteros , Glicerol/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Oocitos/citología , Oocitos/metabolismo , Presión Osmótica , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/agonistas , Ribonucleoproteínas/metabolismo , Estrés FisiológicoRESUMEN
Large cytoplasmic ribonucleoprotein germ granule complexes are a common feature in germ cells. In C. elegans these are called P granules and for much of the life-cycle they associate with nuclear pore complexes in germ cells. P granules are rich in proteins that function in diverse RNA pathways. Here we report that the C. elegans homolog of the eIF4E-transporter IFET-1 is required for oogenesis but not spermatogenesis. We show that IFET-1 is required for translational repression of several maternal mRNAs in the distal gonad and functions in conjunction with the broad-scale translational regulators CGH-1, CAR-1 and PATR-1 to regulate germ cell sex determination. Furthermore we have found that IFET-1 localizes to P granules throughout the gonad and in the germ cell lineage in the embryo. Interestingly, IFET-1 is required for the normal ultrastructure of P granules and for the localization of CGH-1 and CAR-1 to P granules. Our findings suggest that IFET-1 is a key translational regulator and is required for normal P granule formation.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/fisiología , Poro Nuclear/metabolismo , Proteínas Represoras/metabolismo , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Cultivadas , Factor 4E Eucariótico de Iniciación/genética , Mutación/genética , Oogénesis/genética , Biosíntesis de Proteínas , Transporte de Proteínas , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/metabolismo , Proteínas Represoras/genética , Homología de Secuencia de Aminoácido , Procesos de Determinación del SexoRESUMEN
The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to characterize the properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and three other translational regulators. We demonstrate that MEX-3 undergoes phase separation and appears to have intrinsic gel-like properties in vitro. We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. The CCT chaperonin and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins. These novel regulators of condensation are also required for the translational repression of maternal mRNA which is essential for oocyte quality and fertility. The identification of this regulatory network may also have implications for understanding the role of hMex3 phase transitions in cancer.
RESUMEN
The regulation of maternal mRNAs is essential for proper oogenesis, the production of viable gametes, and to avoid birth defects and infertility. Many oogenic RNA-binding proteins have been identified with roles in mRNA metabolism, some of which localize to dynamic ribonucleoprotein granules and others that appear dispersed. Here, we use a combination of in vitro condensation assays and the in vivo C. elegans oogenesis model to determine the intrinsic properties of the conserved KH-domain MEX-3 protein and to identify novel regulators of MEX-3 and the Lsm protein, CAR-1. We demonstrate that MEX-3 undergoes liquid-liquid phase separation and appears to have intrinsic gel-like properties in vitro . We also identify novel roles for the CCT chaperonin and actin in preventing ectopic RNA-binding protein condensates in maturing oocytes that appear to be independent of MEX-3 folding. CCT and actin also oppose the expansion of ER sheets that may promote ectopic condensation of RNA-binding proteins that are associated with de-repression of maternal mRNA. This regulatory network is essential to preserve oocyte quality, prevent infertility, and may have implications for understanding the role of hMex3 phase transitions in cancer. Significance statement: The molecular mechanisms that regulate phase transitions of oogenic RNA-binding proteins are critical to elucidate but are not fully understood.We identify novel regulators of RNA-binding protein phase transitions in maturing oocytes that are required to maintain translational repression of maternal mRNAs and oocyte quality.This study is the first to elucidate a regulatory network involving the CCT chaperonin, actin, and the ER for phase transitions of RNA-binding proteins during oogenesis. Our findings for the conserved MEX-3 protein may also be applicable to better understanding the role of hMex3 phase transitions in cancer.
RESUMEN
Protective cellular responses to stress and aging in the germline are essential for perpetuation of a species; however, relatively few studies have focused on how germ cells respond to stress and aging. We have previously shown that large ribonucleoprotein (RNP) complexes assemble in oocytes of Caenorhabditis during extended meiotic arrest or after environmental stress. Here we explore the regulation of these dynamic RNPs and demonstrate their assembly is coordinated with dramatic, nuclear membrane blebbing in oocytes. Our ultrastructural analyses reveal distinct changes in the endoplasmic reticulum, and the first evidence for the assembly of stacked annulate lamellae in Caenorhabditis. We further show several nucleoporins are required for the complete assembly of RNP granules, and a disruption in RNP granule assembly coupled with a low frequency of nuclear blebbing in arrested oocytes negatively impacts embryonic viability. Our observations support a model where nuclear membrane blebbing is required to increase the trafficking of nucleoporins to the cell cortex in stressed or meiotically arrested cells and to facilitate the recruitment of RNA and protein components of RNPs into large complexes. These new insights may have general implications for better understanding how germ cells preserve their integrity when fertilization is delayed and how cells respond to stress.
Asunto(s)
Caenorhabditis/citología , Caenorhabditis/metabolismo , Proteínas del Helminto/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Oocitos/metabolismo , Ribonucleoproteínas/metabolismo , Transporte Activo de Núcleo Celular , Animales , Animales Modificados Genéticamente , Caenorhabditis/genética , Senescencia Celular , Femenino , Respuesta al Choque Térmico , Masculino , Microscopía Electrónica de Transmisión , Modelos Biológicos , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestructura , Oocitos/citología , Estrés FisiológicoRESUMEN
P granules, ribonucleoprotein (RNP) complexes specific to the cytoplasmic side of the nuclear pores of Caenorhabditis elegans germ cells, are implicated in post-transcriptional control of maternally-transcribed mRNAs. Here we show a relationship in C. elegans of Dicer, the riboendonuclease processing enzyme of the RNA interference and microRNA pathways, with GLH-1, a germline-specific RNA helicase and a constitutive component of P granules. Based on results from GST-pull-downs and immunoprecipitations, GLH-1 binds DCR-1 and this binding does not require RNA. Both GLH-1 protein and glh-1 mRNA levels are reduced in the dcr-1(ok247) null mutant background; conversely, a reduction of DCR-1 protein is observed in the glh-1(gk100) deletion strain. Thus, in the C. elegans germline, DCR-1 and GLH-1 are interdependent. In addition, evidence indicates that DCR-1 protein levels, like those of GLH-1, are likely regulated by the Jun N-terminal kinase (JNK), KGB-1. In adult germ cells, DCR-1 is found in uniformly-distributed, small puncta both throughout the cytoplasm and the nucleus, on the inner side of nuclear pores, and associated with P granules. In arrested oocytes, GLH-1 and DCR-1 re-localize to cytoplasmic and cortically-distributed RNP granules and are necessary to recruit other components to these complexes. We predict that the GLH-1/DCR-1 complex may function in the transport, deposition, or regulation of maternally-transcribed mRNAs and their associated miRNAs.
Asunto(s)
Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/fisiología , ARN Helicasas DEAD-box/fisiología , Ribonucleasa III/fisiología , Ribonucleoproteínas/metabolismo , Animales , Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/química , Núcleo Celular/metabolismo , ARN Helicasas DEAD-box/química , Proteínas Quinasas JNK Activadas por Mitógenos/fisiología , MicroARNs/fisiología , Poro Nuclear/fisiología , Oogénesis , Unión Proteica , Interferencia de ARN , Proteínas de Unión al ARN/fisiologíaRESUMEN
One emerging paradigm of cellular organization of RNA and RNA-binding proteins is the formation of membraneless organelles. Examples of membraneless organelles include several types of ribonucleoprotein granules that form via phase separation. A variety of intracellular pH changes and posttranslational modifications, as well as extracellular stresses, can stimulate the condensation of proteins into granules. For example, the assembly of stress granules induced by oxidative stress, osmotic stress, and heat stress has been well characterized in a variety of somatic cell types. In the germ line, similar stress-induced condensation of proteins occurs; however, less is known about the role of phase separation during gamete production. Researchers who study phase transitions often make use of fluorescent reporters to study the dynamics of RNA-binding proteins during live cell imaging. In this report, we demonstrate that common conditions of live-imaging Caenorhabditis elegans can cause an inadvertent stress and trigger phase transitions of RNA-binding proteins. We show that this imaging-associated stress stimulates decondensation of multiple germ granule proteins and condensation of several P-body proteins. Proteins within larger ribonucleoprotein granules in meiotically arrested oocytes do not appear to be as sensitive to the stress as proteins in diakinesis oocytes of young hermaphrodites, with the exception of the germ granule protein PGL-1. Our results have important methodological implications for all researchers using live-cell imaging techniques. The data also suggest that the RNA-binding proteins within large ribonucleoprotein granules of arrested oocytes may have distinct phases, which we characterize in our companion article.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Células Germinativas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismoRESUMEN
The germ line provides an excellent in vivo system to study the regulation and function of RNP granules. Germ granules are conserved germ line-specific RNP granules that are positioned in the Caenorhabditis elegans adult gonad to function in RNA maintenance, regulation, and surveillance. In Caenorhabditis elegans, when oogenesis undergoes extended meiotic arrest, germ granule proteins and other RNA-binding proteins assemble into much larger RNP granules whose hypothesized function is to regulate RNA metabolism and maintain oocyte quality. To gain insight into the function of oocyte RNP granules, in this report, we characterize distinct phases for four protein components of RNP granules in arrested oocytes. We find that the RNA-binding protein PGL-1 is dynamic and has liquid-like properties, while the intrinsically disordered protein MEG-3 has gel-like properties, similar to the properties of the two proteins in small germ granules of embryos. We find that MEX-3 exhibits several gel-like properties but is more dynamic than MEG-3, while CGH-1 is dynamic but does not consistently exhibit liquid-like characteristics and may be an intermediate phase within RNP granules. These distinct phases of RNA-binding proteins correspond to, and may underlie, differential responses to stress. Interestingly, in oocyte RNP granules, MEG-3 is not required for the condensation of PGL-1 or other RNA-binding proteins, which differs from the role of MEG-3 in small, embryonic germ granules. Lastly, we show that the PUF-5 translational repressor appears to promote MEX-3 and MEG-3 condensation into large RNP granules; however, this role may be associated with regulation of oogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Oocitos/metabolismo , ARN/metabolismo , ARN Nucleotidiltransferasas/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Phase separation has emerged as a widespread process of organizing the cytoplasm of diverse eukaryotic cells. In C. elegans oocytes, several RNA binding proteins are condensed into germ granules called P granules. Prior studies studying the phase transitions of RNA binding proteins in response to increased temperature have suggested that PGL-1 decondenses in oocytes in response to heat. Here, we confirm this finding with a new reporter strain and demonstrate the sensitivity of PGL-1 to temperature changes.
RESUMEN
RNA-binding proteins undergo regulated phase transitions in an array of cell types. The phase separation of RNA-binding proteins, and subsequent formation of RNP condensates or granules, occurs during physiological conditions and can also be induced by stress. Some RNP granules have roles in post-transcriptionally regulating mRNAs, and mutations that prevent the condensation of RNA-binding proteins can reduce an organism's fitness. The reversible and multivalent interactions among RNP granule components can result in RNP complexes that transition among diffuse and condensed states, the latter of which can be pathological; for example, in neurons solid RNP aggregates contribute to disease states such as amyotrophic lateral sclerosis (ALS), and the dysregulation of RNP granules in human germ cells may be involved in Fragile X-associated primary ovarian insufficiency. Thus, regulating the assembly of mRNAs and RNA-binding proteins into discrete granules appears to provide important functions at both cellular and physiological levels. Here we review our current understanding of the role of post-translational modifications (PTMs) in regulating the condensation of RNA-binding proteins in the germ line. We compare and contrast the in vitro evidence that methylation inhibits phase separation of RNA binding proteins, with the extent to which these results apply to the in vivo germ line environment of several model systems. We also focus on the role of phosphorylation in modulating the dynamics of RNP granules in the germ line. Finally, we consider the gaps that exist in our understanding of the role of PTMs in regulating germ line RNP granules.
RESUMEN
As Caenorhabditis elegans hermaphrodites age, sperm become depleted, ovulation arrests, and oocytes accumulate in the gonad arm. Large ribonucleoprotein (RNP) foci form in these arrested oocytes that contain RNA-binding proteins and translationally masked maternal mRNAs. Within 65 min of mating, the RNP foci dissociate and fertilization proceeds. The majority of arrested oocytes with foci result in viable embryos upon fertilization, suggesting that foci are not deleterious to oocyte function. We have determined that foci formation is not strictly a function of aging, and the somatic, ceh-18, branch of the major sperm protein pathway regulates the formation and dissociation of oocyte foci. Our hypothesis for the function of oocyte RNP foci is similar to the RNA-related functions of processing bodies (P bodies) and stress granules; here, we show three orthologs of P body proteins, DCP-2, CAR-1 and CGH-1, and two markers of stress granules, poly (A) binding protein (PABP) and TIA-1, appear to be present in the oocyte RNP foci. Our results are the first in vivo demonstration linking components of P bodies and stress granules in the germ line of a metazoan. Furthermore, our data demonstrate that formation of oocyte RNP foci is inducible in non-arrested oocytes by heat shock, osmotic stress, or anoxia, similar to the induction of stress granules in mammalian cells and P bodies in yeast. These data suggest commonalities between oocytes undergoing delayed fertilization and cells that are stressed environmentally, as to how they modulate mRNAs and regulate translation.
Asunto(s)
Caenorhabditis elegans/fisiología , Calor , Hipoxia , Oocitos/fisiología , Ovulación/fisiología , Estrés Oxidativo , Ribonucleoproteínas/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Gránulos Citoplasmáticos/metabolismo , Femenino , Fertilización/fisiología , Masculino , Oocitos/citología , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Ribonucleoproteínas/genética , Espermatozoides/citología , Espermatozoides/metabolismoRESUMEN
The ability to respond to stress is critical to survival for animals. While stress responses have been studied at both organismal and cellular levels, less attention has been given to the effect of stress on the germ line. Effective germ line adaptations to stress are essential to the propagation of a species. Recent studies suggest that germ cells share some cellular responses to stress with somatic cells, including the assembly of RNP granules, but may also have unique requirements. One fundamental difference between oocytes and sperm, as well as most somatic cells, is the long lifespan of oocytes. Since women are born with all of their eggs, oocytes must maintain their cellular quality over decades prior to fertilization. This prolonged meiotic arrest is one type of stress that eventually contributes to decreased fertility in older women. Germ cell responses to nutritional stress and heat stress have also been well-characterized using model systems. Here we review our current understanding of how germ cells respond to stress, with an emphasis on the dynamic assembly of RNP granules that may be adaptive. We compare and contrast stress responses of male gametes with those of female gametes, and discuss how the dynamic cellular remodeling of the germ line can impact the regulation of gene expression. We also discuss the implications of recent in vitro studies of ribonucleoprotein granule assembly on our understanding of germ line responses to stress, and the gaps that remain in our understanding of the function of RNP granules during stress.
RESUMEN
Cell fate specification in the early C. elegans embryo requires the activity of a family of proteins with CCCH zinc-finger motifs. Two members of the family, MEX-5 and MEX-6, are enriched in the anterior of the early embryo where they inhibit the accumulation of posterior proteins. Embryos from mex-5 single-mutant mothers are inviable due to the misexpression of SKN-1, a transcription factor that can specify mesoderm and endoderm. The aberrant expression of SKN-1 causes a loss of hypodermal and neuronal tissue and an excess of pharyngeal muscle, a Mex phenotype (muscle excess). POS-1, a third protein with CCCH motifs, is concentrated in the posterior of the embryo where it restricts the expression of at least one protein to the anterior. We discovered that reducing the dosage of pos-1(+) can suppress the Mex phenotype of mex-5(-) embryos and that POS-1 binds the 3'-UTR of mex-6. We propose that the suppression of the Mex phenotype by reducing pos-1(+) is due to decreased repression of mex-6 translation. Our detailed analyses of these protein functions reveal complex interactions among the CCCH finger proteins and suggest that their complementary expression patterns might be refined by antagonistic interactions among them.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Mutación/genética , Dedos de Zinc/genética , Regiones no Traducidas 3' , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Portadoras/metabolismo , Cromatografía en Gel , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Endodermo , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Mesodermo , Interferencia de ARN , Proteínas de Unión al ARN , Saccharomyces cerevisiae , Homología de Secuencia de Ácido Nucleico , Supresión Genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Complexes of RNA and RNA binding proteins form large-scale supramolecular structures under many cellular contexts. In Caenorhabditis elegans, small germ granules are present in the germ line that share characteristics with liquid droplets that undergo phase transitions. In meiotically-arrested oocytes of middle-aged hermaphrodites, the germ granules appear to aggregate or condense into large assemblies of RNA-binding proteins and maternal mRNAs. Prior characterization of the assembly of large-scale RNP structures via candidate approaches has identified a small number of regulators of phase transitions in the C. elegans germ line; however, the assembly, function, and regulation of these large RNP assemblies remain incompletely understood. To identify genes that promote remodeling and assembly of large RNP granules in meiotically-arrested oocytes, we performed a targeted, functional RNAi screen and identified over 300 genes that regulate the assembly of the RNA-binding protein MEX-3 into large granules. Among the most common GO classes are several categories related to RNA biology, as well as novel categories such as cell cortex, ER, and chromosome segregation. We found that arrested oocytes that fail to localize MEX-3 into cortical granules display reduced oocyte quality, consistent with the idea that the larger RNP assemblies promote oocyte quality when fertilization is delayed. Interestingly, a relatively small number of genes overlap with the regulators of germ granule assembly during normal development, or with the regulators of solid RNP granules in cgh-1 oocytes, suggesting fundamental differences in the regulation of RNP granule phase transitions during meiotic arrest.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Gránulos Citoplasmáticos/genética , Interferencia de ARN , Ribonucleoproteínas/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular/genética , Segregación Cromosómica , Cromosomas/genética , Gránulos Citoplasmáticos/metabolismo , Citoesqueleto/genética , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Femenino , Células Germinativas , Masculino , Oocitos/citología , Oocitos/fisiología , ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Ribonucleoproteínas/genética , Procesos de Determinación del SexoRESUMEN
In a variety of cell types, ribonucleoprotein (RNP) complexes play critical roles in regulating RNA metabolism. The germ line contains RNPs found also in somatic cells, such as processing (P) bodies and stress granules, as well as several RNPs unique to the germ line, including germ granules, nuage, Balbiani bodies, P granules, U bodies, and sponge bodies. Recent advances have identified a conserved response of germ line RNPs to environmental stresses such as nutritional stress and heat shock. The RNPs increase significantly in size based on cytology; their morphology and subcellular localization changes, and their composition changes. These dynamic changes are reversible when stresses diminish, and similar changes occur in response to aging or extended meiotic arrest prior to fertilization of oocytes. Intriguing correlations exist between the dynamics of the RNPs and the microtubule cytoskeleton and its motor proteins, suggesting a possible mechanism for the assembly and dissociation of the large RNP granules. Similarly, coordinated changes of the nuclear membrane and endoplasmic reticulum may also help unravel the regulatory mechanisms of RNP dynamics. Based on their composition, the RNPs are thought to regulate mRNA decay and/or translation, and initial support for some of these roles is now at hand. Ultimately, the question of why RNP remodeling occurs to such a large extent during a variety of stresses and aging remains to be fully answered, but a current attractive hypothesis is that the plasticity promotes the maintenance of oocyte quality.
Asunto(s)
Envejecimiento/fisiología , Gránulos Citoplasmáticos/metabolismo , Oocitos/metabolismo , Estabilidad del ARN/fisiología , Ribonucleoproteínas/metabolismo , Estrés Fisiológico/fisiología , Animales , Humanos , Oocitos/citología , ARN Mensajero/metabolismoRESUMEN
Vitamin D is an essential micronutrient, necessary for human health. To determine if Caenorhabditis elegans (C. elegans) could function as an effective model to study the mechanisms of action of vitamin D, we asked if vitamin D3 affects C. elegans lifespan. Multiple factors positively impact lifespan in this system including dietary restriction and vitamin E. In addition, the C. elegans DAF-12 nuclear hormone receptor is homologous to the vitamin D receptor in humans and is therefore a candidate for a functional vitamin D receptor. It was hypothesized that vitamin D3 supplementation would increase the lifespan of C. elegans in a DAF-12-dependent manner. Dose-response curves were completed, and results indicate that exposure to 1,000 µg/ml vitamin D3 significantly increased the lifespan of wild-type worms by up to 39% (p<0.001). The daf-12 mutants exposed to 1,000 µg/ml vitamin D3 lived significantly longer than daf-12 controls exposed to 0 µg/ml (p<0.001), but among worms exposed to 1,000 µg/ml vitamin D3, wild type lived significantly longer than daf-12 (p<0.01). The data suggest that vitamin D3 can interact with multiple receptors, possibly implicating the NHR family of nuclear hormone receptors related to DAF-12. This research is the first to our knowledge to utilize C. elegans as a model to study the impact of vitamin D3 on longevity and supports the use of this model system to increase our understanding of vitamin D function at the cellular level, its role in cellular health, and its potential medicinal utility in humans.
Asunto(s)
Envejecimiento/metabolismo , Proteínas de Caenorhabditis elegans/agonistas , Caenorhabditis elegans/efectos de los fármacos , Colecalciferol/farmacología , Receptores Citoplasmáticos y Nucleares/agonistas , Envejecimiento/genética , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Relación Dosis-Respuesta a Droga , Genotipo , Longevidad , Modelos Animales , Mutación , Fenotipo , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de TiempoRESUMEN
In a variety of cell types in plants, animals, and fungi, ribonucleoprotein (RNP) complexes play critical roles in regulating RNA metabolism. These RNP granules include processing bodies and stress granules that are found broadly across cell types, as well as RNP granules unique to the germline, such as P granules, polar granules, sponge bodies, and germinal granules. This review focuses on RNP granules localized in oocytes of the major model systems, Caenorhabditis elegans, Drosophila, Xenopus, mouse, and zebrafish. The signature families of proteins within oocyte RNPs include Vasa and other RNA-binding proteins, decapping activators and enzymes, Argonaute family proteins, and translation initiation complex proteins. This review describes the many recent insights into the dynamics and functions of RNP granules, including their roles in mRNA degradation, mRNA localization, translational regulation, and fertility. The roles of the cytoskeleton and cell organelles in regulating RNP granule assembly are also discussed.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Oocitos/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Gránulos Citoplasmáticos/ultraestructura , Citoesqueleto/metabolismo , Humanos , Oocitos/citología , Oocitos/ultraestructura , Orgánulos/metabolismo , Estrés FisiológicoRESUMEN
Within the rhabditid phylogeny of nematodes, the great majority of species are gonochoristic, having evolved as obligate male/female species. In contrast, the well-studied nematode model system, Caenorhabditis elegans, is androdioecious, utilizing a hermaphroditic/male reproductive system. We have previously determined that in the arrested oocytes of old-aged C. elegans hermaphrodites with depleted sperm, large cytoplasmic ribonucleoprotein foci form. The formation of these foci is reversible, as they dissociate within 3 h after a male mates with the hermaphrodite, resupplying it with sperm. The functional significance of these oocyte foci is not known and previously has not been clear for a hermaphroditic species in which oocytes of young adults wait only approximately 23 min to be fertilized. One hypothesis is that the foci function to maintain maternal mRNAs in oocytes while fertilization is delayed. In this paper, we examine four gonochoristic rhabditid species: Caenorhabditis remanei, Caenorhabditis sp. CB5161, Caenorhabditis sp. PS1010, and Rhabditella axei DF5006. We demonstrate that in three of these four species, ovulation arrests in unmated females until mating occurs and large cytoplasmic foci develop in arrested oocytes. The oocyte foci contain nuclear pore proteins and, in C. remanei at least, the RNA-binding protein MEX-3 as well as RNA. We speculate that these foci maintain the integrity of ooctyes, possibly maintaining the stability or translational repression of maternal mRNAs in unmated females. We further speculate that their presence in oocytes of old-aged C. elegans hermaphrodites is due to conservation from an ancestral gonochoristic state.
Asunto(s)
Caenorhabditis/citología , Oocitos/citología , Ribonucleoproteínas/metabolismo , Animales , Femenino , Proteínas de Complejo Poro Nuclear/metabolismo , OvulaciónRESUMEN
The Caenorhabditis elegans pos-1 gene encodes a zinc-finger protein that is required for germline specification during embryogenesis. The maternally provided mRNA is translationally regulated both spatially and temporally during early development. We have cloned orthologs of pos-1 from C. briggsae and C. remanei, two Caenorhabditis species that have diverged from C. elegans by approximately 20-40 million years. Two regions in the 3' untranslated region are highly conserved among all three species. We find that the pos-1 RNA is expressed in the hermaphrodite and female gonads of C. briggsae and C. remanei but POS-1 protein is not detected at high levels in C. briggsae until the 2-cell stage of embryogenesis. The protein expression is restricted to the germline precursors of the embryo. We conclude that pos-1 appears to be translationally regulated in C. briggsae as it is in C. elegans and speculate the conserved 3' UTR sequences may be involved.