RESUMEN
The 3' ends of almost all eukaryotic mRNAs are generated in an essential two-step processing reaction: endonucleolytic cleavage of an extended precursor followed by the addition of a poly(A) tail. By reconstituting the reaction from overproduced and purified proteins, we provide a minimal list of 14 polypeptides that are essential and two that are stimulatory for RNA processing. In a reaction depending on the polyadenylation signal AAUAAA, the reconstituted system cleaves pre-mRNA at a single preferred site corresponding to the one used in vivo. Among the proteins, cleavage factor I stimulates cleavage but is not essential, consistent with its prominent role in alternative polyadenylation. RBBP6 is required, with structural data showing it to contact and presumably activate the endonuclease CPSF73 through its DWNN domain. The C-terminal domain of RNA polymerase II is dispensable. ATP, but not its hydrolysis, supports RNA cleavage by binding to the hClp1 subunit of cleavage factor II with submicromolar affinity.
Asunto(s)
Poliadenilación , Precursores del ARN , Animales , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Mamíferos/genética , Precursores del ARN/genética , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
Alternative cleavage and polyadenylation (APA) is emerging as an important layer of gene regulation. Factors controlling APA are largely unknown. We developed a reporter-based RNAi screen for APA and identified PABPN1 as a regulator of this process. Genome-wide analysis of APA in human cells showed that loss of PABPN1 resulted in extensive 3' untranslated region shortening. Messenger RNA transcription, stability analyses, and in vitro cleavage assays indicated enhanced usage of proximal cleavage sites (CSs) as the underlying mechanism. Using Cyclin D1 as a test case, we demonstrated that enhanced usage of proximal CSs compromises microRNA-mediated repression. Triplet-repeat expansion in PABPN1 (trePABPN1) causes autosomal-dominant oculopharyngeal muscular dystrophy (OPMD). The expression of trePABPN1 in both a mouse model of OPMD and human cells elicited broad induction of proximal CS usage, linked to binding to endogenous PABPN1 and its sequestration in nuclear aggregates. Our results elucidate a novel function for PABPN1 as a suppressor of APA.
Asunto(s)
Proteína II de Unión a Poli(A)/metabolismo , Poliadenilación , Procesamiento Postranscripcional del ARN , Regiones no Traducidas 3' , Animales , Secuencia de Bases , Línea Celular , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Distrofia Muscular Oculofaríngea/genética , Distrofia Muscular Oculofaríngea/metabolismo , Mutación , Proteína II de Unión a Poli(A)/genética , Proteínas de Unión al ARN/metabolismoRESUMEN
Daytime HONO photolysis is an important source of atmospheric hydroxyl radicals (OH). Knowledge of HONO formation chemistry under typical haze conditions, however, is still limited. In the Multiphase chemistry experiment in Fogs and Aerosols in the North China Plain in 2018, we investigated the wintertime HONO formation and its atmospheric implications at a rural site Gucheng. Three different episodes based on atmospheric aerosol loading levels were classified: clean periods (CPs), moderately polluted periods (MPPs) and severely polluted periods (SPPs). Correlation analysis revealed that HONO formation via heterogeneous conversion of NO2 was more efficient on aerosol surfaces than on ground, highlighting the important role of aerosols in promoting HONO formation. Daytime HONO budget analysis indicated a large missing source (with an average production rate of 0.66 ± 0.26, 0.97 ± 0.47 and 1.45 ± 0.55 ppbV/hr for CPs, MPPs and SPPs, respectively), which strongly correlated with photo-enhanced reactions (NO2 heterogeneous reaction and particulate nitrate photolysis). Average OH formation derived from HONO photolysis reached up to (0.92 ± 0.71), (1.75 ± 1.26) and (1.82 ± 1.47) ppbV/hr in CPs, MPPs and SPPs respectively, much higher than that from O3 photolysis (i.e., (0.004 ± 0.004), (0.006 ± 0.007) and (0.0035 ± 0.0034) ppbV/hr). Such high OH production rates could markedly regulate the atmospheric oxidation capacity and hence promote the formation of secondary aerosols and pollutants.
Asunto(s)
Contaminantes Ambientales , Ácido Nitroso , Ácido Nitroso/análisis , Contaminantes Ambientales/análisis , Dióxido de Nitrógeno/análisis , China , Aerosoles/análisisRESUMEN
Accurate determination of acidity (pH) and ion activities in aqueous droplets is a major experimental and theoretical challenge for understanding and simulating atmospheric multiphase chemistry. Here, we develop a ratiometric Raman spectroscopy method to measure the equilibrium concentration of sulfate (SO42-) and bisulfate (HSO4-) in single microdroplets levitated by aerosol optical tweezers. This approach enables determination of ion activities and pH in aqueous sodium bisulfate droplets under highly supersaturated conditions. The experimental results were compared against aerosol thermodynamic model calculations in terms of simulating aerosol ion concentrations, ion activity coefficients, and pH. We found that the Extended Aerosol Inorganics Model (E-AIM) can well reproduce the experimental results. The alternative model ISORROPIA, however, exhibits substantial deviations in SO42- and HSO4- concentrations and up to a full unit of aerosol pH under acidic conditions, mainly due to discrepancies in simulating ion activity coefficients of SO42--HSO4- equilibrium. Globally, this may cause an average deviation of ISORROPIA from E-AIM by 25 and 65% in predicting SO42- and HSO4- concentrations, respectively. Our results show that it is important to determine aerosol pH and ion activities in the investigation of sulfate formation and related aqueous phase chemistry.
Asunto(s)
Sulfatos , Óxidos de Azufre , Aerosoles/química , Concentración de Iones de Hidrógeno , Espectrometría Raman/métodos , AguaRESUMEN
Nitrous acid (HONO) is an important component of the global nitrogen cycle and can regulate the atmospheric oxidative capacity. Soil is an important source of HONO. [HONO]*, the equilibrium gas-phase concentration over the aqueous solution of nitrous acid in the soil, has been suggested as a key parameter for quantifying soil fluxes of HONO. However, [HONO]* has not yet been well-validated and quantified. Here, we present a method to retrieve [HONO]* by conducting controlled dynamic chamber experiments with soil samples applied with different HONO concentrations at the chamber inlet. We show a bi-directional soil-atmosphere exchange of HONO and confirm the existence of [HONO]* over soil: when [HONO]* is higher than the atmospheric HONO concentration, HONO will be released from soil; otherwise, HONO will be deposited. We demonstrate that [HONO]* is a soil characteristic, which is independent of HONO concentrations in the chamber but varies with different soil water contents. We illustrate the robustness of using [HONO]* for quantifying soil fluxes of HONO, whereas the laboratory-determined chamber HONO fluxes can largely deviate from those in the real world for the same soil sample. This work advances the understanding of the soil-atmosphere exchange of HONO and the evaluation of its impact on the atmospheric oxidizing capacity.
Asunto(s)
Ácido Nitroso , Suelo , Atmósfera , Ciclo del Nitrógeno , Microbiología del Suelo , AguaRESUMEN
Cleavage and polyadenylation specificity factor (CPSF) is the central component of the 3' processing machinery for polyadenylated mRNAs in metazoans: CPSF recognizes the polyadenylation signal AAUAAA, providing sequence specificity in both pre-mRNA cleavage and polyadenylation, and catalyzes pre-mRNA cleavage. Here we show that of the seven polypeptides that have been proposed to constitute CPSF, only four (CPSF160, CPSF30, hFip1, and WDR33) are necessary and sufficient to reconstitute a CPSF subcomplex active in AAUAAA-dependent polyadenylation, whereas CPSF100, CPSF73, and symplekin are dispensable. WDR33 is required for binding of reconstituted CPSF to AAUAAA-containing RNA and can be specifically UV cross-linked to such RNAs, as can CPSF30. Transcriptome-wide identification of WDR33 targets by photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP) showed that WDR33 binds in and very close to the AAUAAA signal in vivo with high specificity. Thus, our data indicate that the large CPSF subunit participating in recognition of the polyadenylation signal is WDR33 and not CPSF160, as suggested by previous studies.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Proteínas Nucleares/metabolismo , Procesamiento de Término de ARN 3'/genética , Factor de Especificidad de Desdoblamiento y Poliadenilación/genética , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Poliadenilación , Unión Proteica/genética , Subunidades de Proteína/metabolismoRESUMEN
Fine-particle pollution associated with winter haze threatens the health of more than 400 million people in the North China Plain. The Multiphase chemistry experiment in Fogs and Aerosols in the North China Plain (McFAN) investigated the physicochemical mechanisms leading to haze formation with a focus on the contributions of multiphase processes in aerosols and fogs. We integrated observations on multiple platforms with regional and box model simulations to identify and characterize the key oxidation processes producing sulfate, nitrate and secondary organic aerosols. An outdoor twin-chamber system was deployed to conduct kinetic experiments under real atmospheric conditions in comparison to literature kinetic data from laboratory studies. The experiments were spanning multiple years since 2017 and an intensive field campaign was performed in the winter of 2018. The location of the site minimizes fast transition between clean and polluted air masses, and regimes representative for the North China Plain were observed at the measurement location in Gucheng near Beijing. The consecutive multi-year experiments document recent trends of PM2.5 pollution and corresponding changes of aerosol physical and chemical properties, enabling in-depth investigations of established and newly proposed chemical mechanisms of haze formation. This study is mainly focusing on the data obtained from the winter campaign 2018. To investigate multiphase chemistry, the results are presented and discussed by means of three characteristic cases: low humidity, high humidity and fog. We find a strong relative humidity dependence of aerosol chemical compositions, suggesting an important role of multiphase chemistry. Compared with the low humidity period, both PM1 and PM2.5 show higher mass fraction of secondary inorganic aerosols (SIA, mainly as nitrate, sulfate and ammonium) and secondary organic aerosols (SOA) during high humidity and fog episodes. The changes in aerosol composition further influence aerosol physical properties, e.g., with higher aerosol hygroscopicity parameter κ and single scattering albedo SSA under high humidity and fog cases. The campaign-averaged aerosol pH is 5.1 ± 0.9, of which the variation is mainly driven by the aerosol water content (AWC) concentrations. Overall, the McFAN experiment provides new evidence of the key role of multiphase reactions in regulating aerosol chemical composition and physical properties in polluted regions.
RESUMEN
Aqueous extracts of biogenic secondary organic aerosols (BSOAs) have been found to exhibit fluorescence that may interfere with the laser/light-induced fluorescence (LIF) detection of primary biological aerosol particles (PBAPs). In this study, we quantified the interference of BSOAs to PBAPs by directly measuring airborne BSOA particles, rather than aqueous extracts. BSOAs were generated by the reaction of d-limonene (LIM) or α-pinene (PIN) and ozone (O3) with or without ammonia in a chamber under controlled conditions. With an excitation wavelength of 355 nm, BSOAs exhibited peak emissions at 464-475 nm, while fungal spores exhibited peak emissions at 460-483 nm; the fluorescence intensity of BSOAs with diameters of 0.7 µm was in the same order of magnitude as that of fungal spores with diameters of 3 µm. The number fraction of 0.7 µm BSOAs that exhibited fluorescence above the threshold was in the range of 1.9-15.9%, depending on the species of precursors, relative humidity (RH), and ammonia. Similarly, the number fraction of 3 µm fungal spores that exhibited fluorescence above the threshold was 4.9-36.2%, depending on the species of fungal spores. Normalized fluorescence by particle volumes suggests that BSOAs exhibited fluorescence in the same order of magnitude as pollen and 10-100 times higher than that of fungal spores. A comparison with ambient particles suggests that BSOAs caused significant interference to ambient fine particles (15 of 16 ambient fine particle measurements likely detected BSOAs) and the interference was smaller for ambient coarse particles (4 of 16 ambient coarse particle measurements likely detected BSOAs) when using LIF instruments.
Asunto(s)
Contaminantes Atmosféricos , Ozono , Aerosoles/análisis , Contaminantes Atmosféricos/análisis , Limoneno , Tamaño de la Partícula , Espectrometría de Fluorescencia , Esporas FúngicasRESUMEN
Cleavage factor II (CF II) is a poorly characterized component of the multiprotein complex catalyzing 3' cleavage and polyadenylation of mammalian mRNA precursors. We have reconstituted CF II as a heterodimer of hPcf11 and hClp1. The heterodimer is active in partially reconstituted cleavage reactions, whereas hClp1 by itself is not. Pcf11 moderately stimulates the RNA 5' kinase activity of hClp1; the kinase activity is dispensable for RNA cleavage. CF II binds RNA with nanomolar affinity. Binding is mediated mostly by the two zinc fingers in the C-terminal region of hPcf11. RNA is bound without pronounced sequence-specificity, but extended G-rich sequences appear to be preferred. We discuss the possibility that CF II contributes to the recognition of cleavage/polyadenylation substrates through interaction with G-rich far-downstream sequence elements.
Asunto(s)
Complejos Multiproteicos/química , Proteínas Nucleares/química , Fosfotransferasas/química , Factores de Transcripción/química , Factores de Escisión y Poliadenilación de ARNm/química , Sitios de Unión , Complejos Multiproteicos/genética , Proteínas Nucleares/genética , Fosfotransferasas/genética , Poliadenilación/genética , Unión Proteica , Multimerización de Proteína , Precursores del ARN/química , Precursores del ARN/genética , Homología de Secuencia de Aminoácido , Factores de Transcripción/genética , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The nuclear poly(A) binding protein (PABPN1) has been suggested, on the basis of biochemical evidence, to play a role in mRNA polyadenylation by strongly increasing the processivity of poly(A) polymerase. While experiments in metazoans have tended to support such a role, the results were not unequivocal, and genetic data show that the S. pombe ortholog of PABPN1, Pab2, is not involved in mRNA polyadenylation. The specific model in which PABPN1 increases the rate of poly(A) tail elongation has never been examined in vivo. Here, we have used 4-thiouridine pulse-labeling to examine the lengths of newly synthesized poly(A) tails in human cells. Knockdown of PABPN1 strongly reduced the synthesis of full-length tails of â¼250 nucleotides, as predicted from biochemical data. We have also purified S. pombe Pab2 and the S. pombe poly(A) polymerase, Pla1, and examined their in vitro activities. Whereas PABPN1 strongly increases the activity of its cognate poly(A) polymerase in vitro, Pab2 was unable to stimulate Pla1 to any significant extent. Thus, in vitro and in vivo data are consistent in supporting a role of PABPN1 but not S. pombe Pab2 in the polyadenylation of mRNA precursors.
Asunto(s)
Poli A/genética , Proteína I de Unión a Poli(A)/genética , Proteínas de Unión a Poli(A)/genética , Polinucleotido Adenililtransferasa/genética , Precursores del ARN/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Regulación de la Expresión Génica , Células HEK293 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Poli A/biosíntesis , Proteína I de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Poliadenilación , Polinucleotido Adenililtransferasa/metabolismo , Precursores del ARN/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Especificidad de la Especie , Especificidad por SustratoRESUMEN
Characteristic particle size, fluorescence intensity, and fluorescence spectra are important features to detect and categorize bioaerosols. A prototype size-resolved single-particle fluorescence spectrometer (S2FS) was developed to simultaneously measure aerodynamic diameters and fluorescence spectra. Emission spectra are dispersed in 512 channels from 370 to 610 nm, where a major portion of biological fluorescence emission occurs. The S2FS consists of an aerodynamic particle sizer and a fluorescence spectrometer with a 355 nm laser excitation source and an intensified charge-coupled device as the detector. Highly fluorescent particles, such as Ambrosia artemisiifolia pollen and Olea europaea pollen, can be distinguished by the S2FS on a single-particle level. For weakly fluorescent particles, fluorescence spectra can only be obtained by averaging multiple particles (between 100 and 3000) of the same kind. Preliminary ambient measurements in Mainz (Germany, central Europe) show that an emission peak at â¼440 nm was frequently observed for fluorescent fine particles (0.5-1 µm). Fluorescent fine particles accounted for 2.8% on average based on the number fraction in the fine mode. Fluorescent coarse particles (>1 µm) accounted for 8.9% on average based on the number fraction, with strongest occurrence observed during a thunderstorm and in the morning.
Asunto(s)
Laboratorios , Aerosoles , Europa (Continente) , Alemania , Tamaño de la Partícula , Espectrometría de FluorescenciaRESUMEN
Oculopharyngeal muscular dystrophy is a late-onset disease caused by an elongation of a natural 10-alanine segment within the N-terminal domain of the nuclear poly(A)-binding protein 1 (PABPN1) to maximally 17 alanines. The disease is characterized by intranuclear deposits consisting primarily of PABPN1. In previous studies, we could show that the N-terminal domain of PABPN1 forms amyloid-like fibrils. Here, we analyze fibril formation of full-length PABPN1. Unexpectedly, fibril formation was independent of the presence of the alanine segment. With regard to fibril formation kinetics and resistance against denaturants, fibrils formed by full-length PABPN1 had completely different properties from those formed by the N-terminal domain. Fourier transformed infrared spectroscopy and limited proteolysis showed that fibrillar PABPN1 has a structure that differs from native PABPN1. Circumstantial evidence is presented that the C-terminal domain is involved in fibril formation.
Asunto(s)
Distrofia Muscular Oculofaríngea/genética , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Deficiencias en la Proteostasis/genética , Alanina/química , Amiloidosis/genética , Amiloidosis/metabolismo , Escherichia coli/genética , Humanos , Distrofia Muscular Oculofaríngea/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Pliegue de Proteína , Estructura Terciaria de Proteína , Deficiencias en la Proteostasis/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Espectrofotometría InfrarrojaRESUMEN
Asymmetric dimethylation of arginine side chains is a common post-translational modification of eukaryotic proteins, which serves mostly to regulate protein-protein interactions. The modification is catalyzed by type I protein arginine methyltransferases, PRMT1 being the predominant member of the family. Determinants of substrate specificity of these enzymes are poorly understood. The Nuclear poly(A) binding protein 1 (PABPN1) is methylated by PRMT1 at 13 arginine residues located in RXR sequences in the protein's C-terminal domain. We have identified a preferred site for PRMT1-catalyzed methylation in PABPN1 and in a corresponding synthetic peptide. Variants of these substrates were analyzed by steady-state kinetic analysis and mass spectrometry. The data indicate that initial methylation is directed toward the preferred arginine residue by an N-terminally adjacent proline. Enhanced methylation upon peptide cyclization suggests that induction of a reverse turn structure is the basis for the ability of the respective proline residue to enable preferred methylation of the neighboring arginine residue, and this notion is supported by far-UV circular dichroism spectroscopy. We suggest that the formation of a reverse turn facilitates the access of arginine side chains to the active sites of PRMT1, which are located in the central cavity of a doughnut-shaped PRMT1 homodimer.
Asunto(s)
Péptidos/química , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Biocatálisis , Humanos , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteína II de Unión a Poli(A)/química , Proteína II de Unión a Poli(A)/genética , Prolina , Ratas , Especificidad por SustratoRESUMEN
The nuclear poly(A) binding protein, PABPN1, promotes mRNA polyadenylation in the cell nucleus by increasing the processivity of poly(A) polymerase and contributing to poly(A) tail length control. In its C-terminal domain, the protein carries 13 arginine residues that are all asymmetrically dimethylated. The function of this modification in PABPN1 has been unknown. Part of the methylated domain serves as nuclear localization signal, binding the import receptor transportin. Here we report that arginine methylation weakens the affinity of PABPN1 for transportin. Recombinant, unmethylated PABPN1 binds more strongly to transportin than its methylated counterpart from mammalian tissue, and in vitro methylation reduces the affinity. Transportin and RNA compete for binding to PABPN1. Methylation favors RNA binding. Transportin also inhibits in vitro methylation of the protein. Finally, a peptide corresponding to the nuclear localization signal of PABPN1 competes with transportin-dependent nuclear import of the protein in a permeabilized cell assay and does so less efficiently when it is methylated. We hypothesize that transportin binding might delay methylation of PABPN1 until after nuclear import. In the nucleus, arginine methylation may favor the transition of PABPN1 to the competing ligand RNA and serve to reduce the risk of the protein being reexported to the cytoplasm by transportin.
Asunto(s)
Arginina/metabolismo , Núcleo Celular/metabolismo , Carioferinas/metabolismo , Proteína II de Unión a Poli(A)/metabolismo , Proteínas de Unión a Poli(A)/metabolismo , Receptores Citoplasmáticos y Nucleares/metabolismo , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bovinos , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Metilación , Datos de Secuencia Molecular , Señales de Localización Nuclear/metabolismo , Proteína II de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/química , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , ARN/metabolismo , Proteínas Recombinantes/metabolismoRESUMEN
Poly(A) tails of mRNAs are synthesized in the cell nucleus with a defined length, approximately 250 nucleotides in mammalian cells. The same type of length control is seen in an in vitro polyadenylation system reconstituted from three proteins: poly(A) polymerase, cleavage and polyadenylation specificity factor (CPSF), and the nuclear poly(A)-binding protein (PABPN1). CPSF, binding the polyadenylation signal AAUAAA, and PABPN1, binding the growing poly(A) tail, cooperatively stimulate poly(A) polymerase such that a complete poly(A) tail is synthesized in one processive event, which terminates at a length of approximately 250 nucleotides. We report that PABPN1 is required to restrict CPSF binding to the AAUAAA sequence and to permit the stimulation of poly(A) polymerase by AAUAAA-bound CPSF to be maintained throughout the elongation reaction. The stimulation by CPSF is disrupted when the poly(A) tail has reached a length of approximately 250 nucleotides, and this terminates processive elongation. PABPN1 measures the length of the tail and is responsible for disrupting the CPSF-poly(A) polymerase interaction.
Asunto(s)
Factor de Especificidad de Desdoblamiento y Poliadenilación/metabolismo , Poli A/metabolismo , Proteína I de Unión a Poli(A)/metabolismo , Polinucleotido Adenililtransferasa/metabolismo , Animales , Bovinos , Ensayo de Cambio de Movilidad Electroforética , Poli A/química , Unión Proteica , Señales de Poliadenilación de ARN 3'/genéticaRESUMEN
The heterodimeric HIF (hypoxia-inducible factor)-1 is a transcriptional master regulator of several genes involved in mammalian oxygen homoeostasis. Besides the well described regulation of the HIF-1alpha subunit via hydroxylation-mediated protein stability in hypoxia, there are several indications of an additional translational control of the HIF-1alpha mRNA, especially after growth factor stimulation. We identified an interaction of CPEB (cytoplasmic polyadenylation-element-binding protein) 1 and CPEB2 with the 3'-UTR (untranslated region) of HIF-1alpha mRNA. Overexpression of CPEB1 and CPEB2 affected HIF-1alpha protein levels mediated by the 3'-UTR of HIF-1alpha mRNA. Stimulation of neuroblastoma SK-N-MC cells with insulin and thus activation of endogenous CPEBs increased the expression of a luciferase reporter gene fused to the 3'-UTR of HIF-1alpha as well as endogenous HIF-1alpha protein levels. This could be abrogated by treating the cells with CPEB1 or CPEB2 siRNAs (short interfering RNAs). Injection of HIF-1alpha cRNA into Xenopus oocytes verified the elongation of the poly(A)+ (polyadenylated) tail by cytoplasmic polyadenylation. Thus CPEB1 and CPEB2 are involved in the regulation of HIF-1alpha following insulin stimulation.
Asunto(s)
Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Regiones no Traducidas 3'/genética , Animales , Monóxido de Carbono/farmacología , Femenino , Técnica del Anticuerpo Fluorescente , Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Luciferasas/genética , Luciferasas/metabolismo , Ratones , Modelos Biológicos , Oocitos/metabolismo , Poliadenilación/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Biosíntesis de Proteínas/efectos de los fármacos , ARN Mensajero/genética , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/fisiología , Xenopus , Factores de Escisión y Poliadenilación de ARNm/genética , Factores de Escisión y Poliadenilación de ARNm/fisiologíaRESUMEN
Poly (A) tails are found at the 3' ends of almost all eukaryotic mRNAs. They are bound by two different poly (A) binding proteins, PABPC in the cytoplasm and PABPN1 in the nucleus. PABPC functions in the initiation of translation and in the regulation of mRNA decay. In both functions, an interaction with the m7G cap at the 5' end of the message plays an important role. PABPN1 is involved in the synthesis of poly (A) tails, increasing the processivity of poly (A) polymerase and contributing to defining the length of a newly synthesized poly (A) tail.
Asunto(s)
Proteínas de Unión a Poli(A)/química , Proteínas de Unión a Poli(A)/fisiología , Animales , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas Fúngicas/fisiología , Humanos , Modelos Biológicos , Proteína I de Unión a Poli(A)/fisiología , Proteína II de Unión a Poli(A)/fisiología , Unión Proteica , ARN Mensajero/metabolismo , Relación Estructura-Actividad , TemperaturaRESUMEN
Hydrogen sulphide emission in lichens as a response to low concentration SO2 , fumigation was investigated. In an open flow-through system several lichen species were fumigated with 36 ppb SO2 , Two species were also fumigated with higher concentrations (72, 119, 122 and 198 ppb SO2 ,). Hydrogen sulphide emission was monitored concurrently by cryogenic trapping and analysis with gas chromatography. All tested species increased H, S emission significantly in response to fumigation with 36 ppb SO2 . Parmelina tiliacea (L.) Hale and Cladina rangiferina (L.) Wigg. released significantly more H2 S (0.098±0.015 and 0.073±0.013 pmol H2 S g-1 d. wt s-1 , respectively) than Parmelina quercina (Ach.) Hale, Ramalina menziesii Tayl. and Parmelia sulcata Tayl. (0.028 ± 0.01, 0.025±0.014 and 0.023±0.013 pmol H2 S g-1 d. wt s-1 , respectively). Release of H2 S in Hypogymnia physodes was enhanced by increasing SO2 concentrations up to 72 ppb SO2 . No significant difference in H2 S emission in the dark vs. in the light was found. Generally, no correlation was found between photosynthetic activity and H2 S emission for the tested species. Uptake of SO2 was similar for all species, at 24.7 ± 5.6 pmol SO2 g-1 d. wt s-1 in 36 ppb SO2 and increasing at greater SO2 concentrations. Therefore, H2 S-S release represents only 0.11-0.74% of SO2 -S uptake.
RESUMEN
Research studies aimed at advancing cancer prevention, diagnosis, and treatment depend on a number of key resources, including a ready supply of high-quality annotated biospecimens from diverse ethnic populations that can be used to test new drugs, assess the validity of prognostic biomarkers, and develop tailor-made therapies. In November 2011, KHCCBIO was established at the King Hussein Cancer Center (KHCC) with the support of Seventh Framework Programme (FP7) funding from the European Union (khccbio.khcc.jo). KHCCBIO was developed for the purpose of achieving an ISO accredited cancer biobank through the collection, processing, and preservation of high-quality, clinically annotated biospecimens from consenting cancer patients, making it the first cancer biobank of its kind in Jordan. The establishment of a state-of-the-art, standardized biospecimen repository of matched normal and lung tumor tissue, in addition to blood components such as serum, plasma, and white blood cells, was achieved through the support and experience of its European partners, Trinity College Dublin, Biostór Ireland, and accelopment AG. To date, KHCCBIO along with its partners, have worked closely in establishing an ISO Quality Management System (QMS) under which the biobank will operate. A Quality Policy Manual, Validation, and Training plan have been developed in addition to the development of standard operating procedures (SOPs) for consenting policies on ethical issues, data privacy, confidentiality, and biobanking bylaws. SOPs have also been drafted according to best international practices and implemented for the donation, procurement, processing, testing, preservation, storage, and distribution of tissues and blood samples from lung cancer patients, which will form the basis for the procurement of other cancer types. KHCCBIO will be the first ISO accredited cancer biobank from a diverse ethnic Middle Eastern and North African population. It will provide a unique and valuable resource of high-quality human biospecimens and anonymized clinicopathological data to the cancer research communities world-wide.
Asunto(s)
Bancos de Muestras Biológicas/normas , Investigación Biomédica , Neoplasias , Humanos , Jordania , Medio OrienteRESUMEN
Chemical crosslinking in combination with mass spectrometry has matured into an alternative approach to derive low-resolution structural information of proteins and protein complexes. Yet, one of the major drawbacks of this strategy remains the lack of software that is able to handle the large MS datasets that are created after chemical crosslinking and enzymatic digestion of the crosslinking reaction mixtures. Here, we describe a software, termed StavroX, which has been specifically designed for analyzing highly complex crosslinking datasets. The StavroX software was evaluated for three diverse biological systems: (1) the complex between calmodulin and a peptide derived from Munc13, (2) an N-terminal ß-laminin fragment, and (3) the complex between guanylyl cyclase activating protein-2 and a peptide derived from retinal guanylyl cyclase. We show that the StavroX software is advantageous for analyzing crosslinked products due to its easy-to-use graphical user interface and the highly automated analysis of mass spectrometry (MS) and tandem mass spectrometry (MS/MS) data resulting in short times for analysis. StavroX is expected to give a further push to the chemical crosslinking approach as a routine technique for protein interaction studies.