RESUMO
Following the transcription step, the newly synthesized mRNA is exported from the nucleus to the cytoplasm and further to the translation site. The TREX-2 complex is involved in the step of mRNA export from the nucleus to the cytoplasm. This complex in Drosophila melanogaster consists of four proteins: Xmas-2, PCID2, ENY2, and Sem1p. In our work, we have shown that deletion of the C-terminal sequence of PCID2 leads to a decrease in the interaction of the protein with RNA and to impaired mRNA export from the nucleus to the cytoplasm in D. melanogaster.
Assuntos
Núcleo Celular , Drosophila melanogaster , Animais , Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The TREX-2 protein complex is the key complex involved in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a joint protein complex of TREX-2 with ORC was isolated in D. melanogaster. It was shown that the interaction of TREX-2 with ORC is necessary for efficient mRNA export from the nucleus to the cytoplasm. In this work, we showed that the TREX-2-ORC joint complex is also formed in human cells.
Assuntos
Drosophila melanogaster , Proteínas Nucleares , Animais , Humanos , Transporte Ativo do Núcleo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila melanogaster/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Nucleocytoplasmic exchange in the cell occurs through the nuclear pore complexes (NPCs). NPCs are large multiprotein complexes with octagonal symmetry about their axis and imperfect mirror symmetry about a plane parallel with the nuclear envelop (NE). NPC fuses the inner and outer nuclear membranes and opens up a channel between nucleus and cytoplasm. NPC is built of nucleoporins. Each nucleoporin occurs in at least eight copies per NPC. Inside the NPC a permeability barrier forms by which NPCs can provide fast and selectable transport of molecules from one side of the nuclear membrane to the other. NPC architecture is based on hierarchical principle of organization. Nucleoporins are integrated into complexes that oligomerizes into bigger octomeric high-order structures. These structures are the main components of NPCs. In the first part of this work, the main attention is paid to NPC structure and nucleoporin properties. The second part is dedicated to mechanisms of NPC assembly and disassembly at different stages of the cell cycle.
Assuntos
Complexo de Proteínas Formadoras de Poros Nucleares , Poro Nuclear , Poro Nuclear/química , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/análise , Complexo de Proteínas Formadoras de Poros Nucleares/química , Membrana Nuclear/metabolismo , Citoplasma/metabolismo , Complexos Multiproteicos/análise , Complexos Multiproteicos/metabolismo , Transporte Ativo do Núcleo CelularRESUMO
The TREX-2 complex integrates several stages of gene expression, such as transcriptional activation and mRNA export. In D. melanogaster, TREX-2 consists of four major proteins: Xmas-2, ENY2, PCID2, and Sem1p. The Xmas-2 protein is the core subunit of the complex, with which other TREX-2 subunits interact. Xmas-2 homologues were found in all higher eukaryotes. Previously, it was shown that the human Xmas-2 homologue, GANP protein, can undergo cleavage into two parts, probably during apoptosis. We showed that the Xmas-2 protein of D. melanogaster can also split into two fragments. The resulting fragments of the protein correspond to the two large Xmas-2 domains. Protein splitting is observed both in vivo and in vitro. However, Xmas-2 cleavage in D. melanogaster is observed under normal conditions and is probably a part of the mechanism of transcription and mRNA export regulation in D. melanogaster.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Humanos , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/metabolismoRESUMO
The Drosophila melanogaster Maleless (MLE) protein is a conserved helicase involved in a wide range of gene expression regulation processes. A MLE ortholog, named DHX9, was found in many higher eukaryotes, including humans. DHX9 is involved in diverse processes, such as genome stability maintenance, replication, transcription, splicing, editing and transport of cellular and viral RNAs, and translation regulation. Some of these functions are understood in detail today, while most of them remain uncharacterized. Study of the functions of the MLE ortholog in mammals in vivo is limited by the fact that the loss of function of this protein is lethal at the embryonic stage. In D. melanogaster, helicase MLE was originally discovered and studied for a long time as a participant in dosage compensation. Recent evidence indicates that helicase MLE is involved in the same cell processes in D. melanogaster and mammals and that many of its functions are evolutionarily conserved. Experiments in D. melanogaster revealed new important MLE functions, such as a role in hormone-dependent regulation of transcription and interactions with the SAGA transcription complex, other transcriptional cofactors, and chromatin remodeling complexes. Unlike in mammals, MLE mutations do not cause embryonic lethality in D. melanogaster, and the MLE functions are possible to study in vivo throughout ontogenesis in females and up to the pupal stage in males. The human MLE ortholog is a potential target for anticancer and antiviral therapies. Further investigation of the MLE functions in D. melanogaster is therefore of both basic and applied importance. The review discusses the systematic position, domain structure, and conserved and specific functions of MLE helicase in D. melanogaster.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Animais , Feminino , Humanos , Masculino , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona , DNA Helicases/genética , DNA Helicases/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Eucariotos/genética , Regulação da Expressão Gênica , Mamíferos/genética , Fatores de Transcrição/genética , Cromossomo X/metabolismoRESUMO
We examined postoperative material from 28 patients aged 39-61 years with gliomas of different degrees of anaplasia (the diagnosis was histologically verified according to the WHO classification of CNS tumors) who had not previously received antitumor treatment. In glioma tissue, the glucose concentration was significantly higher than in the brain tissue of subjects dead from traumas (control), while lactate concentration did not differ from that in the control group or was lower. Hexokinase activity demonstrated a tendency to an increase in grade I and significant elevation in grades II and III, while in grade IV gliomas, this parameter did not differ from the control. Activities of the pentose-phosphate pathway enzymes glucose-6-phosphate dehydrogenase and transketolase increased with increasing of tumor anaplasia. Activity of glycogen synthase 3ß kinase was significantly higher than in the control group. IDH1 mutation was discovered in 40% cases, the MGMT promoter methylation was detected in more than 50%, the Ki-67 level increased with increasing tumor anaplasia. The most significant correlations with glioma markers were detected for glucose-6-phosphate dehydrogenase and glycogen synthase 3ß kinase. Activities of the studied enzymes of carbohydrate metabolism significantly correlated with Ki-67 marker.
Assuntos
Química Encefálica/fisiologia , Neoplasias Encefálicas/patologia , Glioma/genética , Glioma/patologia , Glucosefosfato Desidrogenase/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Adulto , Anaplasia/patologia , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Metabolismo dos Carboidratos/genética , Metabolismo dos Carboidratos/fisiologia , Metilação de DNA/genética , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/genética , Glucose/análise , Hexoquinase/metabolismo , Humanos , Isocitrato Desidrogenase/genética , Ácido Láctico/análise , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Transcetolase/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
SAGA, the multicomponent complex responsible for acetylation of histone N-terminal lysine residues, is involved in the transcription activation of a wide range of eukaryote genes. SAGA contains a protein module, DUB, which is responsible for histone deubiquitination. In this paper we show that the DUB module may be found within cells independently of SAGA. In the absence of" SAGA, the DUB module may be recruited to the promoters of Pol III-transcribed genes, but not to the Pol II-dependent promoters. The DUB module is required to recruit transcription factor Brfl, a subunit of the Pol III-recruiting TFIIIB complex, to the promoters of Pol III-dependent genes. The DUB-module interacts with Pol III in vivo. The DUB-module is essential for recruiting both TFIIIB complexes and PBP complexes to the promoters of Pol III-dependent genes.
Assuntos
RNA Polimerase II , Fatores de Transcrição , Regulação da Expressão Gênica , Histonas/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
The TREX-2 protein complex is the key participant in the export of mRNA from the nucleus to the cytoplasm through the nuclear pores. Previously, a protein complex of D. melanogaster consisting of TREX-2 and ORC complexes was purified. It was shown that, in the TREX-2-ORC complex, the Xmas-2 protein, which is the platform for TREX-2 assembly, interacts with the Orc3 protein. The aim of this work was to investigate what regions of the Xmas-2 amino acid sequence are involved in the interaction with Orc3. It was shown that the interaction of Xmas-2 with Orc3 requires a C-terminal region of Xmas-2 located downstream of the CID domain.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Drosophila melanogaster/genética , Domínios e Motivos de Interação entre Proteínas , Transporte de RNA , RNA Mensageiro/genética , Homologia de Sequência de AminoácidosRESUMO
The TREX-2 complex is essential for the general nuclear mRNA export in eukaryotes. TREX-2 interacts with the nuclear pore and transcriptional apparatus and links transcription to the mRNA export. However, it remains poorly understood how the TREX-2-dependent nuclear export is connected to the subsequent stages of mRNA trafficking. Here, we show that the PCID2 subunit of Drosophila TREX-2 is present in the cytoplasm of the cell. The cytoplasmic PCID2 directly interacts with the NudC protein and this interaction maintains its stability in the cytoplasm. Moreover, PCID2 is associated with the cytoplasmic mRNA and microtubules. The PCID2 knockdown blocks nuclear export of mRNA and also affects the general mRNA transport into the cytoplasm. These data suggest that PCID2 could be the link between the nuclear TREX-2-dependent export and the subsequent cytoplasmic trafficking of mRNA.
Assuntos
Transporte Ativo do Núcleo Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , RNA Mensageiro/metabolismo , Animais , Núcleo Celular/genética , Citoplasma/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , RNA Mensageiro/genéticaRESUMO
TREX-2 complex is responsible for general mRNA export from nucleus to cytoplasm in eukaryote. The main protein of TREX-2 complex of D. melanogaster is protein Xmas-2. Its homologues in yeast and humans are Sac3 and GANP proteins, respectively. All three proteins contain the highly conserved domain Sac3-GANP, which is essential for interaction of TREX-2 complex with mRNA and another protein of the complex, PCID2. We identified two Xmas-2 homologues in D. melanogaster using the Sac3-GANP family domain sequence. These proteins have a common domain responsible for interaction with the PCID2 protein and RNA and are present in other eukaryotes. The function of these proteins is unknown. However, on the basis of their structural organization, we can assume that they interact with nucleic acids.
Assuntos
Biologia Computacional/métodos , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Exodesoxirribonucleases/metabolismo , Transporte de RNA/fisiologia , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Animais , Sequência Conservada , Bases de Dados Genéticas , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Exodesoxirribonucleases/genética , Transporte de RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Homologia de SequênciaRESUMO
The general snRNA gene transcription apparatus has been extensively studied. However, the role of coactivators in this process is far from being clearly understood. Here, we have demonstrated that the Drosophila SAGA complex interacts with the PBP complex, the key component of the snRNA gene transcription apparatus, and is present at the promoter regions of the snRNA genes transcribed by both the RNA polymerase II and RNA polymerase III (U6 snRNA). We show that SAGA interacts with the Brf1 transcription factor, which is a part of the RNA polymerase III transcription apparatus and is present at promoters of a number of Pol III-transcribed genes. Mutations inactivating several SAGA subunit genes resulted in reduced snRNA levels in adult flies, indicating that SAGA is indeed the transcriptional coactivator for the snRNA genes. The transcription of the Pol II and Pol III-transcribed U genes was reduced by mutations in all tested SAGA complex subunits. Therefore, the transcription of the Pol II and Pol III-transcribed U genes was reduced by the mutations in the deubiquitinase module, as well as in the acetyltransferase module of the SAGA, indicating that the whole complex is essential for their transcription. Therefore, the SAGA complex activates snRNA genes suggesting its wide involvement in the regulation of gene transcription, and consequently, in the maintenance of cellular homeostasis.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/genética , Regulação da Expressão Gênica , Complexos Multiproteicos/fisiologia , RNA Nuclear Pequeno/genética , Fatores de Transcrição/fisiologia , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Mutação , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , RNA Polimerase III/metabolismo , Fator de Transcrição TFIIIB/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
It is shown that Drosophila melanogaster has Xmas mRNA whose alternative splicing leads to the formation of three transcripts: Xmas, Xmas-2, and Xmas-1. As a result, three proteins are synthesized: Xmas, Xmas-2, and, presumably, Xmas-1. The size of the Xmas protein is close to the size of its homologue in humans. Adult flies contain large amounts of this protein, whereas in embryos it is absent.
Assuntos
Processamento Alternativo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Animais , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
RNA-binding proteins play an important role in RNA metabolism, especially in mRNA biogenesis and subsequent expression patterns regulation. RNA immunoprecipitation (RIP) is a powerful tool for detecting protein-RNA associations. In this paper, we briefly cover the history of this method for analyzing RNA-protein interactions and reviewing a number of modifications of the RIP technique. We also present an adjusted RIP protocol that was modified for Drosophila S2 cell culture. The use of this protocol allows one to perform the efficient precipitation of RNA-protein complexes and harvest RNA in amounts that are sufficient for its downstream analysis.
Assuntos
Drosophila melanogaster/genética , Imunoprecipitação , Proteínas de Ligação a RNA/isolamento & purificação , RNA/isolamento & purificação , Animais , Linhagem Celular , Drosophila melanogaster/citologiaRESUMO
The molecular mechanisms that coordinate transcription, processing, mRNP assembly, and mRNA export from the nucleus through nuclear pores into the cytoplasm have been the focus of intense research in recent years. Data demonstrating a tight association between the processes involved in gene expression are considered. The main protein complexes that play a role in mRNA export are described. The complexes are recruited to mRNA at steps preceding the mRNA export. The functions that the complexes perform at particular steps of gene expression are analyzed, and protein complexes responsible for quality control of mRNP discussed.
Assuntos
Núcleo Celular/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , RNA Mensageiro/metabolismo , Transporte Ativo do Núcleo Celular/fisiologia , Animais , Núcleo Celular/genética , Humanos , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , RNA Mensageiro/genéticaRESUMO
mRNA transport from the nucleus to the cytoplasm is an essential step of eukaryotic gene expression. A pre-mRNA molecule undergoes modification, such as 5'-capping, splicing, and 3'-end processing, in the nucleus. The molecule being modified interacts with a large number of proteins and, thus, mRNP particles are formed. The binding of factors involved in nuclear export also occurs during transcription and mRNA processing. We have shown that the functioning of TREX-2, an mRNA export complex, is restricted to the nucleus. We used the method of RNA coprecipitation that enables the selective extraction of RNA-protein complexes from samples to show that the transcription elongation complex TREX interacts with mRNA of the ß-tubulin 56D gene over the entire length of the molecule. The capping protein Cbp80 reacted both with the cap structure and with a specific part of the coding mRNA of the ß-tubulin 56D gene. The TREX-2 complex that mediates mRNA export from the nucleus to the cytoplasm is bound to the same part of the coding sequence. Thus, we identified a common binding site for all of the complexes under investigation on the mRNA of ß-tubulin 56D. Co-immunoprecipitation reactions performed with S2 cell extracts revealed interactions between the components of complexes involved in transcription elongation, maturation, and export of mRNA. The model of molecular folding for the mRNP particle involving the mRNA of ß-tubulin 56D has been proposed.
Assuntos
Modelos Biológicos , Complexos Multiproteicos/metabolismo , RNA Mensageiro/metabolismo , Ribonucleoproteínas/metabolismo , Elongação da Transcrição Genética/fisiologia , Tubulina (Proteína)/metabolismo , Animais , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Complexos Multiproteicos/genética , Complexo Proteico Nuclear de Ligação ao Cap/genética , Complexo Proteico Nuclear de Ligação ao Cap/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Tubulina (Proteína)/genéticaRESUMO
RNA-binding proteins (RBPs) play an important role in regulating gene expression at the posttranscriptional level, including the steps of pre-mRNA splicing, polyadenylation, mRNA stabilization, mRNA export from the nucleus to the cytoplasm, mRNA localization, and translation. RBPs regulate these processes primarily by binding to specific sequence elements in newly synthesized or mature transcripts. While many RPBs are known to recognize certain nucleotide sequences in RNA, information is insufficient for others. In particular, RBPs often compete for RNA binding or interact with RNA cooperatively. Hence, it is of importance to study the RNA-protein interactions in vivo. Numerous methods have been developed to identify the target nucleotide sequences of RBPs. The methods include the electrophoretic mobility shift assay (EMSA), systematic evolution of ligands by exponential enrichment (SELEX), RNA pull-down assay, RNA footprinting, RNA immunoprecipitation (RIP), UV-induced crosslinking immunoprecipitation (CLIP) and its variants, and measurement of the level for newly synthesized transcripts. Each of the methods has its limitation, and several methods supplementing each other should be employed in order to detect the RNA sequence to which a protein binds.
Assuntos
Ensaio de Desvio de Mobilidade Eletroforética , Precursores de RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Técnica de Seleção de Aptâmeros , Animais , Sítios de Ligação , Regulação da Expressão Gênica , Humanos , Imunoprecipitação/métodos , Ligação Proteica , Precursores de RNA/química , Precursores de RNA/genética , RNA Mensageiro/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Análise de Sequência de RNA/métodosRESUMO
Eukaryotic gene expression is known as a multistep process of high complexity. Transcription is one of cardinal and tightly regulated phase during gene expression. To provide accurate and precise work of gene regulation apparatus including a plethora of modification of chromatin structure and nucleosome dynamic turnover must be occurred. All transcription steps are under control of large multiprotein coactivator complexes. In this review we discuss an evolutionary conservative SAGA complex, which acetylates and deubiquitinates histones during transcription activation and furthermore is involved in subsequent stages of mRNP biogenesis and export.
Assuntos
Regulação da Expressão Gênica , Complexos Multiproteicos/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Transcrição Gênica , Acetilação , Histonas/genética , Histonas/metabolismo , Complexos Multiproteicos/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/imunologia , Saccharomyces cerevisiae/genética , UbiquitinaçãoRESUMO
SAGA is a histone acetyltransferase complex, that cotranscriptionally performs histone modifications and is implicated in regulation of gene expression at the level of changes in chromatin structure. SAGA is also involved in mRNP biogenesis and export. In this review, we examined a contribution of SAGA and its subunits in the development. We also discuss the diseases associated with impaired activity of SAGA subunits.
