RESUMEN
NEW FINDINGS: What is the central question of this study? Gonadal hormones modulate cerebrovascular function while insulin-like growth factor 1 (IGF-1) facilitates exercise-mediated cerebral angiogenesis; puberty is a critical period of neurodevelopment alongside elevated gonadal hormone and IGF-1 activity: but whether exercise training across puberty enhances cerebrovascular function is unkown. What is the main finding and its importance? Cerebral blood flow is elevated in endurance trained adolescent males when compared to untrained counterparts. However, cerebrovascular reactivity to hypercapnia is faster in trained vs. untrained children, but not adolescents. Exercise-induced improvements in cerebrovascular function are attainable as early as the first decade of life. ABSTRACT: Global cerebral blood flow (gCBF) and cerebrovascular reactivity to hypercapnia ( CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ ) are modulated by gonadal hormone activity, while insulin-like growth factor 1 facilitates exercise-mediated cerebral angiogenesis in adults. Whether critical periods of heightened hormonal and neural development during puberty represent an opportunity to further enhance gCBF and CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ is currently unknown. Therefore, we used duplex ultrasound to assess gCBF and CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ in n = 128 adolescents characterised as endurance-exercise trained (males: n = 30, females: n = 36) or untrained (males: n = 29, females: n = 33). Participants were further categorised as pre- (males: n = 35, females: n = 33) or post- (males: n = 24, females: n = 36) peak height velocity (PHV) to determine pubertal or 'maturity' status. Three-factor ANOVA was used to identify main and interaction effects of maturity status, biological sex and training status on gCBF and CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ . Data are reported as group means (SD). Pre-PHV youth demonstrated elevated gCBF and slower CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ mean response times than post-PHV counterparts (both: P ≤ 0.001). gCBF was only elevated in post-PHV trained males when compared to untrained counterparts (634 (43) vs. 578 (46) ml min-1 ; P = 0.007). However, CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ mean response time was faster in pre- (72 (20) vs. 95 (29) s; P ≤ 0.001), but not post-PHV (P = 0.721) trained youth when compared to untrained counterparts. Cardiorespiratory fitness was associated with gCBF in post-PHV youth (r2 = 0.19; P ≤ 0.001) and CV R C O 2 ${\mathrm{CV}}{{\mathrm{R}}_{{\mathrm{C}}{{\mathrm{O}}_{\mathrm{2}}}}}$ mean response time in pre-PHV youth (r2 = 0.13; P = 0.014). Higher cardiorespiratory fitness during adolescence can elevate gCBF while exercise training during childhood primes the development of cerebrovascular function, highlighting the importance of exercise training during the early stages of life in shaping the cerebrovascular phenotype.
Asunto(s)
Hipercapnia , Factor I del Crecimiento Similar a la Insulina , Masculino , Adulto , Niño , Femenino , Humanos , Adolescente , Ejercicio Físico/fisiología , Circulación Cerebrovascular/fisiología , Hormonas GonadalesRESUMEN
Hypertension is an important risk factor for age-related cognitive decline and neuronal pathologies. Studies have shown a correlation between hypertension, disruption in neurovascular coupling and cerebral autoregulation, and cognitive decline. However, the mechanisms behind this are unclear. To further understand this, it is advantageous to study neurovascular coupling as hypertension progresses in a rodent model. Here, we use a longitudinal functional MRI (fMRI) protocol to assess the impact of hypertension on neurovascular coupling in spontaneously hypertensive rats (SHRs). Eight female SHRs were studied at 2, 4, and 6 months of age, as hypertension progressed. Under an IV infusion of propofol, animals underwent fMRI, functional MR spectroscopy, and cerebral blood flow (CBF) quantification to study changes in neurovascular coupling over time. Blood pressure significantly increased at 4 and 6 months (P < .0001). CBF significantly increased at 4 months old (P < .05), in the acute stage of hypertension. The size of the active region decreased significantly at 6 months old (P < .05). Change in glutamate signal during activation, and N-acetyl-aspartate (NAA) signal, remained constant. This study shows that, while cerebral autoregulation is impaired in acute hypertension, the blood oxygenation-level-dependent (BOLD) response remains unaltered until later stages. At this stage, the consistent NAA and glutamate signals show that neuronal death has not occurred, and that neuronal activity is not affected at this stage. This suggests that neuronal activity and viability is not lost until much later, and changes observed here in BOLD activity are due to vascular effects.
Asunto(s)
Encéfalo/diagnóstico por imagen , Circulación Cerebrovascular/fisiología , Hipertensión/diagnóstico por imagen , Imagen por Resonancia Magnética/métodos , Acoplamiento Neurovascular/fisiología , Animales , Presión Sanguínea/fisiología , Encéfalo/irrigación sanguínea , Femenino , Hipertensión/fisiopatología , Ratas , Ratas Endogámicas SHRRESUMEN
As the population ages, the incidence of age-related neurological diseases and cognitive decline increases. To further understand disease-related changes in brain function it is advantageous to examine brain activity changes in healthy aging rodent models to permit mechanistic investigation. Here, we examine the suitability, in rodents, of using a novel, minimally invasive anaesthesia protocol in combination with a functional MRI protocol to assess alterations in neuronal activity due to physiological aging. 11 Wistar Han female rats were studied at 7, 9, 12, 15 and 18 months of age. Under an intravenous infusion of propofol, animals underwent functional magnetic resonance imaging (fMRI) and functional magnetic resonance spectroscopy (fMRS) with forepaw stimulation to quantify neurotransmitter activity, and resting cerebral blood flow (CBF) quantification using arterial spin labelling (ASL) to study changes in neurovascular coupling over time. Animals showed a significant decrease in size of the active region with age (P â< â0.05). fMRS results showed a significant decrease in glutamate change with stimulation (ΔGlu) with age (P â< â0.05), and ΔGlu became negative from 12 months onwards. Global CBF remained constant for the duration of the study. This study shows age related changes in the blood oxygen level dependent (BOLD) response in rodents that correlate with those seen in humans. The results also suggest that a reduction in synaptic glutamate turnover with age may underlie the reduction in the BOLD response, while CBF is preserved.
Asunto(s)
Envejecimiento/fisiología , Encéfalo/fisiología , Modelos Animales , Neuroimagen/métodos , Anestésicos Intravenosos/farmacología , Animales , Encéfalo/efectos de los fármacos , Femenino , Estudios Longitudinales , Imagen por Resonancia Magnética/métodos , Espectroscopía de Resonancia Magnética/métodos , Propofol/farmacología , Ratas , Ratas WistarRESUMEN
Disability and effectiveness of physical therapy are highly variable following ischemic stroke due to different brain regions being affected. Functional magnetic resonance imaging (fMRI) studies of patients in the months and years following stroke have given some insight into how the brain recovers lost functions. Initially, new pathways are recruited to compensate for the lost region, showing as a brighter blood oxygen-level-dependent (BOLD) signal over a larger area during a task than in healthy controls. Subsequently, activity is reduced to baseline levels as pathways become more efficient, mimicking the process of learning typically seen during development. Preclinical models of ischemic stroke aim to enhance understanding of the biology underlying recovery following stroke. However, the pattern of recruitment and focusing seen in humans has not been observed in preclinical fMRI studies that are highly variable methodologically. Resting-state fMRI studies show more consistency; however, there are still confounding factors to address. Anesthesia and method of stroke induction are the two main sources of variability in preclinical studies; improvements here can reduce variability and increase the intensity and reproducibility of the BOLD response detected by fMRI. Differences in task or stimulus and differences in analysis method also present a source of variability. This review compares clinical and preclinical fMRI studies of recovery following stroke and focuses on how refinement of preclinical models and MRI methods may obtain more representative fMRI data in relation to human studies.
Asunto(s)
Isquemia Encefálica/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Recuperación de la Función/fisiología , Accidente Cerebrovascular/diagnóstico por imagen , Humanos , Imagen por Resonancia Magnética/métodos , Reproducibilidad de los ResultadosRESUMEN
The transport of rice glutelin storage proteins to the storage vacuoles requires the Rab5 GTPase and its related guanine nucleotide exchange factor (Rab5-GEF). Loss of function of these membrane vesicular trafficking factors results in the initial secretion of storage proteins and later their partial engulfment by the plasma membrane to form an extracellular paramural body (PMB), an aborted endosome complex. Here, we show that in the rice Rab5-GEF mutant glup6, glutelin RNAs are specifically mislocalized from their normal location on the cisternal endoplasmic reticulum (ER) to the protein body-ER, and are also apparently translocated to the PMBs. We substantiated the association of mRNAs with this aborted endosome complex by RNA-seq of PMBs purified by flow cytometry. Two PMB-associated groups of RNA were readily resolved: those that were specifically enriched in this aborted complex and those that were highly expressed in the cytoplasm. Examination of the PMB-enriched RNAs indicated that they were not a random sampling of the glup6 transcriptome but, instead, encompassed only a few functional mRNA classes. Although specific autophagy is also an alternative mechanism, our results support the view that RNA localization may co-opt membrane vesicular trafficking, and that many RNAs that share function or intracellular location are co-transported in developing rice seeds.
Asunto(s)
Glútenes/genética , Factores de Intercambio de Guanina Nucleótido/genética , Oryza/genética , Proteínas de Plantas/genética , ARN Mensajero/genética , ARN de Planta/genética , Proteínas de Unión al GTP rab5/genética , Glútenes/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , ARN Mensajero/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
RNA binding proteins (RBPs) play an important role in mRNA metabolism including synthesis, maturation, transport, localization, and stability. In developing rice seeds, RNAs that code for the major storage proteins are transported to specific domains of the cortical endoplasmic reticulum (ER) by a regulated mechanism requiring RNA cis-localization elements, or zipcodes. Putative trans-acting RBPs that recognize prolamine RNA zipcodes required for restricted localization to protein body-ER have previously been identified. Here, we describe the identification of RBP-P using a Northwestern blot approach as an RBP that recognizes and binds to glutelin zipcode RNA, which is required for proper RNA localization to cisternal-ER. RBP-P protein expression coincides with that of glutelin during seed maturation and is localized to both the nucleus and cytosol. RNA-immunoprecipitation and subsequent RT-PCR analysis further demonstrated that RBP-P interacts with glutelin RNAs. In vitro RNA-protein UV-crosslinking assays showed that recombinant RBP-P binds strongly to glutelin mRNA, and in particular, 3' UTR and zipcode RNA. RBP-P also exhibited strong binding activity to a glutelin intron sequence, suggesting that RBP-P might participate in mRNA splicing. Overall, these results support a multifunctional role for RBP-P in glutelin mRNA metabolism, perhaps in nuclear pre-mRNA splicing and cytosolic localization to the cisternal-ER.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/fisiología , Glútenes/metabolismo , Oryza/metabolismo , Transporte de Proteínas/fisiología , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Secuencia de Aminoácidos , Animales , Glútenes/genética , Datos de Secuencia Molecular , Oryza/genética , Unión Proteica , ARN de Planta/genética , Proteínas de Unión al ARN/genética , Semillas/genética , Semillas/metabolismoRESUMEN
RNAs for the storage proteins, glutelins and prolamines, contain zipcode sequences, which target them to specific subdomains of the cortical endoplasmic reticulum in developing rice (Oryza sativa) seeds. Fifteen RNA binding proteins (RBPs) specifically bind to the prolamine zipcode sequences and are likely to play an important role in the transport and localization of this storage protein RNA. To understand the underlying basis for the binding of multiple protein species to the prolamine zipcode sequences, the relationship of five of these RBPs, RBP-A, RBP-I, RBP-J, RBP-K, and RBP-Q, were studied. These five RBPs, which belong to the heterogeneous nuclear ribonucleoprotein class, bind specifically to the 5' coding regions as well as to the 3' untranslated region zipcode RNAs but not to a control RNA sequence. Coimmunoprecipitation-immunoblot analyses in the presence or absence of ribonuclease showed that these five RBPs are assembled into three multiprotein complexes to form at least two zipcode RNA-protein assemblies. One cytoplasmic-localized zipcode assembly contained two multiprotein complexes sharing a common core consisting of RBP-J and RBP-K and either RBP-A (A-J-K) or RBP-I (I-J-K). A second zipcode assembly of possibly nuclear origin consists of a multiprotein complex containing RBP-Q and modified forms of the other protein complexes. These results suggest that prolamine RNA transport is initiated in the nucleus to form a zipcode-protein assembly, which is remodeled in the cytoplasm to target the RNA to its proper location on the cortical endoplasmic reticulum.
Asunto(s)
Complejos Multiproteicos/metabolismo , Oryza/metabolismo , Fenilpropanolamina/metabolismo , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Regiones no Traducidas 3'/genética , Secuencia de Bases , Bioensayo , Biotinilación , Fluorescencia , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Inmunoprecipitación , Solanum lycopersicum/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oryza/embriología , Oryza/genética , Filogenia , Unión Proteica , Protoplastos/metabolismo , ARN de Planta/genética , Ribonucleasas/metabolismo , Semillas/genética , Semillas/crecimiento & desarrollo , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Técnicas del Sistema de Dos HíbridosRESUMEN
RNA binding proteins (RBPs) play an important role not only in nuclear gene expression, but also in cytosolic events, including RNA transport, localization, translation, and stability. Although over 200 RBPs are predicted from the Arabidopsis genome alone, relatively little is known about these proteins in plants as many exhibit no homology to known RBPs in other eukaryotes. Furthermore, RBPs likely have low expression levels making them difficult to identify and study. As part of our continuing efforts to understand plant cytosolic gene expression and the factors involved, we employed a combination of affinity chromatography and proteomic techniques to enrich for low abundance RBPs in developing rice seed. Our results have been compiled into RiceRBP (http://www.bioinformatics2.wsu.edu/RiceRBP), a database that contains 257 experimentally identified proteins, many of which have not previously been predicted to be RBPs. For each of the identified proteins, RiceRBP provides information on transcript and protein sequence, predicted protein domains, details of the experimental identification, and whether antibodies have been generated for public use. In addition, tools are available to analyze expression patterns for the identified genes, view phylogentic relationships and search for orthologous proteins. RiceRBP is a valuable tool for the community in the study of plant RBPs.
RESUMEN
Rice (Oryza sativa) glutelins are synthesized on the endoplasmic reticulum as larger precursors, which are then transported via the Golgi to the protein storage vacuole (PSV), where they are processed into acidic and basic subunits. Three independent glutelin precursor mutant4 (glup4) rice lines, which accumulated elevated levels of proglutelin over the wild type, were identified as loss-of-function mutants of Rab5a, the small GTPase involved in vesicular membrane transport. In addition to the plasma membrane, Rab5a colocalizes with glutelins on the Golgi apparatus, Golgi-derived dense vesicles, and the PSV, suggesting that Rab5a participates in the transport of the proglutelin from the Golgi to the PSV. This spatial distribution pattern was dramatically altered in the glup4 mutants. Numerous smaller protein bodies containing glutelin and α-globulin were evident, and the proteins were secreted extracellularly. Moreover, all three independent glup4 allelic lines displayed the novel appearance of a large dilated, structurally complex paramural body containing proglutelins, α-globulins, membrane biomarkers for the Golgi apparatus, prevacuolar compartment, PSV, and the endoplasmic reticulum luminal chaperones BiP and protein disulfide isomerase as well as ß-glucan. These results indicate that the formation of the paramural bodies in glup4 endosperm was due to a significant disruption of endocytosis and membrane vesicular transport by Rab5a loss of function. Overall, Rab5a is required not only for the intracellular transport of proglutelins from the Golgi to the PSV in rice endosperm but also in the maintenance of the general structural organization of the endomembrane system in developing rice seeds.
Asunto(s)
Endospermo/crecimiento & desarrollo , Glútenes/metabolismo , Aparato de Golgi/metabolismo , Oryza/enzimología , Oryza/crecimiento & desarrollo , Vacuolas/metabolismo , Proteínas de Unión al GTP rab5/metabolismo , alfa-Globulinas/metabolismo , Endosomas/metabolismo , Endospermo/enzimología , Membranas Intracelulares/metabolismo , Mutación , Oryza/genética , Oryza/metabolismo , Precursores de Proteínas/metabolismo , Transporte de Proteínas , Proteoglicanos/metabolismo , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Proteínas de Unión al GTP rab5/genéticaRESUMEN
RNA-binding proteins play critical roles at multiple steps during gene expression, including mRNA transport and translation. mRNA transport is particularly important in rice (Oryza sativa L.) in order to ensure the proper localization of the prolamine and glutelin seed storage proteins. However, relatively little information is available about RNA-binding proteins that have been isolated or characterized in plants. The RiceRBP database is a novel resource for the analysis of RNA-binding proteins in rice. RiceRBP contains 257 experimentally identified RNA-binding proteins, which are derived from at least 221 distinct rice genes. Many of the identified proteins catalogued in RiceRBP had not previously been annotated or predicted to bind RNA. RiceRBP provides tools to facilitate the analysis of the identified RNA-binding proteins, including information about predicted protein domains, phylogenetic relationships, and expression patterns of the identified genes. Importantly, RiceRBP also contains tools to search and analyze predicted RNA-binding protein orthologs in other plant species. We anticipate that the data and analysis tools provided by RiceRBP should facilitate the study of plant RNA-binding proteins. RiceRBP is available at http://www.bioinformatics2.wsu.edu/RiceRBP.
Asunto(s)
Bases de Datos Genéticas , Bases de Datos de Proteínas , Oryza/genética , Proteínas de Unión al ARN/análisis , Proteínas de Unión al ARN/genética , Biología Computacional , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genoma de Planta , Hordeum/genética , Hordeum/metabolismo , Almacenamiento y Recuperación de la Información/métodos , Internet , Espectrometría de Masas , Análisis de Secuencia por Matrices de Oligonucleótidos , Oryza/metabolismo , Filogenia , Proteínas de Plantas/análisis , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Terciaria de Proteína , ARN de Planta/metabolismo , Proteínas de Unión al ARN/metabolismo , Semillas/genética , Semillas/metabolismo , Programas Informáticos , Sorghum/genética , Sorghum/metabolismo , Triticum/genética , Triticum/metabolismo , Interfaz Usuario-Computador , Zea mays/genética , Zea mays/metabolismoRESUMEN
The rice esp2 mutation was previously characterized by the abnormal accumulation of elevated levels of proglutelin and the absence of an endosperm-specific protein disulfide isomerase like (PDIL1-1). Here we show that Esp2 is the structural gene for PDIL1-1 and that this lumenal chaperone is asymmetrically distributed within the cortical endoplasmic reticulum (ER) and largely restricted to the cisternal ER. Temporal studies indicate that PDIL1-1 is essential for the maturation of proglutelin only when its rate of synthesis significantly exceeds its export from the ER, a condition resulting in its build up in the ER lumen and the induction of ER quality control processes which lower glutelin levels as well as those of the other storage proteins. As proglutelin is initially synthesized on the cisternal ER, its deposition within prolamine protein bodies in esp2 suggests that PDIL1-1 helps retain proglutelin in the cisternal ER lumen until it attains competence for ER export and, thereby, indirectly preventing heterotypic interactions with prolamine polypeptides.
Asunto(s)
Retículo Endoplásmico/metabolismo , Endospermo/metabolismo , Glútenes/metabolismo , Oryza/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Proteínas de Almacenamiento de Semillas/metabolismo , Dosificación de Gen , Oryza/genética , Proteína Disulfuro Isomerasas/genética , Proteínas de Almacenamiento de Semillas/genética , Análisis de Secuencia de ADNRESUMEN
The messenger RNA of the rice seed storage protein prolamine is targeted to the endoplasmic reticulum (ER) membranes surrounding prolamine protein bodies via a mechanism, which is dependent upon both RNA sorting signals and the actin cytoskeleton. In this study we have used an RNA bait corresponding to the previously characterized 5'CDS prolamine cis-localization sequence for the capture of RNA binding proteins (RBPs) from cytoskeleton-enriched fractions of developing rice seed. In comparison to a control RNA, the cis-localization RNA bait sequence led to the capture of a much larger number of proteins, 18 of which have been identified by tandem mass spectrometry. Western blots demonstrate that several of the candidate proteins analyzed to date show good to excellent specificity for binding to cis-localization sequences over the control RNA bait. Temporal expression studies showed that steady state protein levels for one RNA binding protein, RBP-A, paralleled prolamine gene expression. Immunoprecipitation studies showed that RBP-A is bound to prolamine and glutelin RNAs in vivo, supporting a direct role in storage protein gene expression. Using confocal immunofluorescence microscopy, RBP-A was found to be distributed to multiple compartments in the cell. In addition to the nucleus, RBP-A co-localizes with microtubules and is associated with cortical ER membranes. Collectively, these results indicate that employing a combination of in vitro binding and in vivo binding and localization studies is a valid strategy for the identification of putative prolamine mRNA binding proteins, such as RBP-A, which play a role in controlling expression of storage protein mRNAs in the cytoplasm.
Asunto(s)
Citoesqueleto/metabolismo , Oryza/embriología , Proteínas de Plantas/metabolismo , Prolaminas/metabolismo , Proteínas de Unión al ARN/aislamiento & purificación , Proteínas de Unión al ARN/metabolismo , Semillas/metabolismo , Secuencia de Bases , Reactivos de Enlaces Cruzados/farmacología , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/efectos de la radiación , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Glútenes/genética , Glútenes/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/efectos de la radiación , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Microtúbulos/efectos de la radiación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sistemas de Lectura Abierta/genética , Oryza/efectos de los fármacos , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/aislamiento & purificación , Prolaminas/genética , Unión Proteica , Transporte de ARN/efectos de los fármacos , Transporte de ARN/efectos de la radiación , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Semillas/efectos de los fármacos , Semillas/genética , Semillas/efectos de la radiación , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Fracciones Subcelulares/efectos de la radiación , Rayos UltravioletaRESUMEN
In eukaryotes, RNA binding proteins (RBPs) play an integral role not only in RNA processing within the nucleus, but also in the cytoplasmic events of RNA transport, localization, translation, storage and degradation. While many studies have been done, relatively little is known about RBPs in plants. As part of our continuing efforts to understand cytoplasmic gene expression events in developing rice seed (Oryza sativa L.), a proteomics approach was used to identify cytoplasmic-localized, cytoskeletal-associated RBPs. The nucleic acid binding fraction from a cytoskeletal-enriched rice seed extract was isolated by Poly(U)-Sepharose affinity chromatography and analyzed using 2D gel electrophoresis. Analysis of 162 excised protein spots using mass spectrometry led to the identification of 148 distinct proteins, in addition to the highly abundant globulin and glutelin seed storage proteins. Identified proteins include those involved in RNA processing, translation, protein modification, cell signaling, and metabolism, as well as a number of hypothetical proteins. Proteins of particular interest with roles in RNA metabolism are discussed. These results have been deposited within the Rice RNA Binding Protein Database as part of an integrated study of plant cytoskeletal-associated RBPs using developing rice seed as a model.
Asunto(s)
Citoesqueleto/metabolismo , Oryza/química , Proteoma/análisis , Proteómica/métodos , Proteínas de Unión al ARN/análisis , Semillas/química , Forma de la Célula , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Metabolismo Energético , Proteínas de Plantas/análisis , Proteínas de Plantas/clasificación , Biosíntesis de Proteínas , ARN de Planta/metabolismo , Transducción de Señal , Estrés FisiológicoRESUMEN
Previous studies have demonstrated that the major storage protein RNAs found in the rice endosperm are transported as particles via actomyosin to specific subdomains of the cortical endoplasmic reticulum. In this study, we examined the potential role of OsTudor-SN, a major cytoskeletal-associated RNA binding protein, in RNA transport and localization. OsTudor-SN molecules occur as high-molecular-weight forms, the integrity of which are sensitive to RNase. Immunoprecipitation followed by RT-PCR showed that OsTudor-SN binds prolamine and glutelin RNAs. Immunofluorescence studies using affinity-purified antibodies show that OsTudor-SNs exists as particles in the cytoplasm, and are distributed to both the protein body endoplasmic reticulum (ER) and cisternal ER. Examination of OsTudor-SN particles in transgenic rice plants expressing GFP-tagged prolamine RNA transport particles showed co-localization of OsTudor-SN and GFP, suggesting a role in RNA transport. Consistent with this view, GFP-tagged OsTudor-SN is observed in living endosperm sections as moving particles, a property inhibited by microfilament inhibitors. Downregulation of OsTudor-SN by antisense and RNAi resulted in a decrease in steady state prolamine RNA and protein levels, and a reduction in the number of prolamine protein bodies. Collectively, these results show that OsTudor-SN is a component of the RNA transport particle, and may control storage protein biosynthesis by regulating one or more processes leading to the transport, localization and anchoring of their RNAs to the cortical ER.
Asunto(s)
Citoplasma/metabolismo , Proteínas de Microtúbulos/fisiología , Oryza/metabolismo , Proteínas de Plantas/fisiología , ARN de Planta/metabolismo , Proteínas de Unión al ARN/fisiología , Regulación de la Expresión Génica de las Plantas , Proteínas Fluorescentes Verdes/análisis , Proteínas de Microtúbulos/antagonistas & inhibidores , Proteínas de Microtúbulos/química , Oryza/genética , Oryza/crecimiento & desarrollo , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Prolaminas , Interferencia de ARN , Transporte de ARN , Proteínas de Unión al ARN/antagonistas & inhibidores , Proteínas de Unión al ARN/química , Proteínas Recombinantes de Fusión/análisis , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismoRESUMEN
Rice synthesizes and accumulates high levels of 2 distinct classes of seed storage proteins and sorts them to separate intracellular compartments, making it an ideal model system for studying the mechanisms of storage protein synthesis, transport, and deposition. In rice, RNA localization dictates the initial site of storage protein synthesis on specific subdomains of the cortical endoplasmic reticulum (ER), and there is a direct relation between the RNA localization site and the final destination of the encoded protein within the endomembrane system. Current data support the existence of 3 parallel RNA localization pathways leading from the nucleus to the actively synthesizing cortical ER. Additional pathways may exist for the synthesis of cytoplasmic and nuclear-encoded proteins targeted to organelles, the latter located in a stratified arrangement in developing endosperm cells. The study of rice mutants, which accumulate unprocessed glutelin precursors, indicates that these multiple pathways prevent nonproductive interactions between different classes of storage proteins that would otherwise disrupt protein sorting. Indeed, it appears that the prevention of disruptive interactions between different classes of storage proteins plays a key role in their biosynthesis in rice. In addition to highlighting the unique features of the plant endomembrane system and describing the relation between RNA and protein localization, this minireview will attempt to address a number of questions raised by recent studies on these processes.