104 research outputs found
Mass spectrometry–based relative quantification of proteins in precatalytic and catalytically active spliceosomes by metabolic labeling (SILAC), chemical labeling (iTRAQ), and label-free spectral count.
No full textThe spliceosome undergoes major changes in protein and RNA composition during pre-mRNA splicing. Knowing the proteins—and their respective quantities—at each spliceosomal assembly stage is critical for understanding the molecular mechanisms and regulation of splicing. Here, we applied three independent mass spectrometry (MS)–based approaches for quantification of these proteins: (1) metabolic labeling by SILAC, (2) chemical labeling by iTRAQ, and (3) label-free spectral count for quantification of the protein composition of the human spliceosomal precatalytic B and catalytic C complexes. In total we were able to quantify 157 proteins by at least two of the three approaches. Our quantification shows that only a very small subset of spliceosomal proteins (the U5 and U2 Sm proteins, a subset of U5 snRNP-specific proteins, and the U2 snRNP-specific proteins U2A′ and U2B′′) remains unaltered upon transition from the B to the C complex. The MS-based quantification approaches classify the majority of proteins as dynamically associated specifically with the B or the C complex. In terms of experimental procedure and the methodical aspect of this work, we show that metabolically labeled spliceosomes are functionally active in terms of their assembly and splicing kinetics and can be utilized for quantitative studies. Moreover, we obtain consistent quantification results from all three methods, including the relatively straightforward and inexpensive label-free spectral count technique
3D cryo-EM structure of an active step I spliceosome and localization of its catalytic core.
No full textThe spliceosome excises introns from pre-mRNA in a two-step splicing reaction. So far, the three-dimensional (3D) structure of a spliceosome with preserved catalytic activity has remained elusive. Here, we determined the 3D structure of the human, catalytically active step I spliceosome (C complex) by cryo-electron microscopy (cryo-EM) in vitrified ice. Via immunolabeling we mapped the position of the 5′ exon. The C complex contains an unusually salt-stable ribonucleoprotein (RNP) core that harbors its catalytic center. We determined the 3D structure of this RNP core and also that of a post-step II particle, the 35S U5 snRNP, which contains most of the C complex core proteins. As C complex domains could be recognized in these structures, their position in the C complex could be determined, thereby allowing the region harboring the spliceosome's catalytic core to be localized
Phosphorylation Drives a Dynamic Switch in Serine/Arginine-Rich Proteins
No full textSummarySerine/arginine-rich (SR) proteins are important players in RNA metabolism and are extensively phosphorylated at serine residues in RS repeats. Here, we show that phosphorylation switches the RS domain of the serine/arginine-rich splicing factor 1 from a fully disordered state to a partially rigidified arch-like structure. Nuclear magnetic resonance spectroscopy in combination with molecular dynamics simulations revealed that the conformational switch is restricted to RS repeats, critically depends on the phosphate charge state and strongly decreases the conformational entropy of RS domains. The dynamic switch also occurs in the 100 kDa SR-related protein hPrp28, for which phosphorylation at the RS repeat is required for spliceosome assembly. Thus, a phosphorylation-induced dynamic switch is common to the class of serine/arginine-rich proteins and provides a molecular basis for the functional redundancy of serine/arginine-rich proteins and the profound influence of RS domain phosphorylation on protein-protein and protein-RNA interactions
Phirutne rromnǎ: lenqëre hakaja, dotǎ aj xod-aktualizàcia
No full textTill now, a state of nomadic Romani women has been described only from a position of an external observer. The author supposes that narrative sources should be used more actively. To actualize this concept he studies recollections of nomadic life, focusing on Romani women’s traditional ways of earning, such as fortune-telling and begging, and their main value – a family. The article shows how Romani women used to risk for husbands and children. They had to take the initiative and to show mother with every day to feed themselves and their relatives. At the same time, married Romani women lived at the mercy of patriarchy. That created the basic paradox of their lives. Gypsy fortune tellers had real economical independence. Outside of families they looked as free women, but inside they uncomplainingly received whipping. The author want to show subjects that are hardly shown in special literature at the moment. Particularly, he writes about the fact that Romani women have actively taken part in a fight against Nazism during the World War II. Another little known subject is women’s literacy. Though most nomadic Romani women couldn’t read, but indeed exclusions existed. It is interesting, too, such a fact as Romani women could get a high social status with an elderly age, so that men take into account their opinion in traditional court. Sometimes, as it can be seen from examples, families were named for female ancestors. The author studies also psychological possibilities used by Gypsy wives to affect their husbands’ decisions.Natalia Gancar
Rekonstrùkcia thaj brakhipen e rromane ʒivipnasqëre aj folkloraqëre
No full textAuthor is a painter. Together with the members of the Romen Theatre in Moscow he carries out a project of theatrical reconstructions of the traditional scenes and situations of the gypsy camp life (dances among others). The acts are prepared according to detailed historical studies. They are subsequently photographed and filmed to form an archival collection to be used by researchers in the future
Si
No full textTaking into account the results of LDA+U+SO-modeling of the electronic structure of Fe1-xCoxSi, temperature-prolonged magnetic phase transitions of the first order in strongly correlated alloys with a broken crystal structure of the B20 type are investigated. It is shown that such phase transitions are induced by concentration and thermodynamic fluctuations for compositions with 0.2<x<0.65 and are accompanied by a change in the sign of the mode-mode coupling parameter. In this case, an intermediate region appears between the long-range order phases and the paramagnetic phase, with the short-range spin order characterized by ferromagnetic and helicoidal spatial fluctuations. We have defined the intervals of external magnetic fields, at which skyrmion microstructures spatially limited by the crossover of helicoidal and ferromagnetic fluctuations appear in the considered region of the extended phase transition. The peculiarities of the temperature dependences of the magnetic susceptibility in an external magnetic field, characteristic of the appearance of skyrmions, are obtained. © 2021 Author(s)
TET dioxygenases localize at splicing speckles and promote RNA splicing
No full textThe dynamic regulation of RNA metabolism plays a crucial part in cellular function, with emerging evidence suggesting an important role for RNA modifications in this process. This study explores the relationship between RNA splicing and the TET dioxygenase activity, shedding light on the role of hm5C (RNA 5-hydroxymethylcytosine), and TET proteins in RNA metabolism. Integrating data from mass spectrometry, AlphaFold structural modeling, microscopic analysis, and different functional assays, including in vitro splicing, TET proteins were found to regulate splicing. We show that TET1, TET2, and TET3 interact with the splicing factors U2AF1 and U2AF2. Interestingly, TET dioxygenases localize in splicing speckles in mammalian and Drosophila cells. TET speckles association was found to be RNA-dependent, but also rely on its interaction with splicing factors. Furthermore, cellular splicing assays revealed that all three TET proteins promote splicing efficiency independent of their catalytic activity. Interestingly, though, the oxidation of m5C to hm5C restores splicing efficiency in vitro. The latter highlights the regulatory role of cytosine modifications in RNA metabolism. These findings provide insights into the complex interplay between RNA modifications and splicing, suggesting a multifaceted role for TET proteins in RNA metabolism beyond their canonical function in the oxidation of 5mC in DNA
Characterization of purified human Bact spliceosomal complexes reveals compositional and morphological changes during spliceosome activation and first step catalysis.
No full textTo better understand the compositional and structural dynamics of the human spliceosome during its activation, we set out to isolate spliceosomal complexes formed after precatalytic B but prior to catalytically active C complexes. By shortening the polypyrimidine tract of the PM5 pre-mRNA, which lacks a 3′ splice site and 3′ exon, we stalled spliceosome assembly at the activation stage. We subsequently affinity purified human Bact complexes under the same conditions previously used to isolate B and C complexes, and analyzed their protein composition by mass spectrometry. A comparison of the protein composition of these complexes allowed a fine dissection of compositional changes during the B to Bact and Bact to C transitions, and comparisons with the Saccharomyces cerevisiae Bact complex revealed that the compositional dynamics of the spliceosome during activation are largely conserved between lower and higher eukaryotes. Human SF3b155 and CDC5L were shown to be phosphorylated specifically during the B to Bact and Bact to C transition, respectively, suggesting these modifications function at these stages of splicing. The two-dimensional structure of the human Bact complex was determined by electron microscopy, and a comparison with the B complex revealed that the morphology of the human spliceosome changes significantly during its activation. The overall architecture of the human and S. cerevisiae Bact complex is similar, suggesting that many of the higher order interactions among spliceosomal components, as well as their dynamics, are also largely conserved.</jats:p
Report of the CCQM-K130 : Nitrogen mass fraction measurements in glycine
No full textMass fraction of nitrogen is very important pointer because the results of these measurements are often used for determination of protein mass fraction that is an important indicator of the quality of the vast majority of food products and raw materials, in particular dry milk powder. Proteins-enzymes catalyze chemical reactions, protein along with fats and carbohydrates is one of the indicators characterizing the energy value of food, so its definition is mandatory for all food products. The aim of this key comparison CCQM-K130 and pilot study P166 is to support National Measurement Institutes (NMIs) and Designated Institutes (DIs) to demonstrate the validity of the procedures the employed for determination of nitrogen mass fraction in glycine. The study material for this key comparison and pilot study has been selected to be representative as one of the aminoacid – the simplest part of the protein. Glycine is an amino acid, single acid that does not have any isomers (melting point –290 °C; specific heat of evaporation − 528,6 J/kg; specific melting heat − 981,1 J/kg; pKa − 2,34, molar mass - 75,07 g/mol, density - 1,607 g/cm3). Ural Scientific Research Institute for Metrology (UNIIM) acted as the coordinating laboratory of this comparison and pilot study. Eight NMIs participated in this key comparison and two NMIs participated in Pilot study. The results of Pilot study are excluded from the Report B.Fil: Medvedevskikh, Maria. Ural Scientific Research Institute for Metrology (UNIIM); RusiaFil: Jury, Bessonov. Ural Scientific Research Institute for Metrology (UNIIM); RusiaFil: Krasheninina, Maria. Ural Scientific Research Institute for Metrology (UNIIM); RusiaFil: Acco Garcia, Steve Ali. (INACAL); PerúFil: Haraldsson, Conny. SP Technical Research Institute of Sweden; SueciaFil: Rodriguez, M. Alejandra. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Rodriguez, Gabriela. Instituto Nacional de Tecnología Industrial (INTI); ArgentinaFil: Salvo, Karino. Laboratorio Tecnológico del Uruguay (LATU); UruguayFil: Gavrilkin, Vladimir. (UkrCSM); UcraniaFil: Kulik, Sergey. (UkrCSM); UcraniaFil: Samuel, Laly. (MSL); Nueva ZelandaFil: do Rego, Eliane C. P. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Wollinger, Wagner. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: Monteiro, Tânia M. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); BrasilFil: de Carvalho, Lucas J. Instituto Nacional de Metrologia, Qualidade e Tecnologia (INMetro); Brasi
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