The nuclear pore complex (NPC) is responsible for transport between the cytoplasm and nucleoplasm and one of the more intricate structures of eukaryotic cells. Typically composed of over 300 polypeptides, the NPC shares evolutionary origins with endo-membrane and intraflagellar transport system complexes. The modern NPC was fully established by the time of the last eukaryotic common ancestor and, hence, prior to eukaryote diversification. Despite the complexity, the NPC structure is surprisingly flexible with considerable variation between lineages. Here, we review diversification of the NPC in major taxa in view of recent advances in genomic and structural characterisation of plant, protist and nucleomorph NPCs and discuss the implications for NPC evolution. Furthermore, we highlight these changes in the context of mRNA export and consider how this process may have influenced NPC diversity. We reveal the NPC as a platform for continual evolution and adaptation.

• Keywords
• eukaryogenesis, evolutionary biology, nuclear pores, nuclear protein transport,
• MeSH
• Biological Evolution * MeSH
• Biological Transport MeSH
• Nuclear Pore metabolism   MeSH
• Membrane Proteins metabolism   MeSH
• RNA, Messenger metabolism   MeSH
• Mitosis MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• Membrane Proteins MeSH
• RNA, Messenger MeSH

Nicotinamide phosphoribosyltransferase (NAMPT) is located in both the nucleus and cytoplasm and has multiple biological functions including catalyzing the rate-limiting step in NAD synthesis. Moreover, up-regulated NAMPT expression has been observed in many cancers. However, the determinants and regulation of NAMPT's nuclear transport are not known. Here, we constructed a GFP-NAMPT fusion protein to study NAMPT's subcellular trafficking. We observed that in unsynchronized 3T3-L1 preadipocytes, 25% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 62% had higher GFP-NAMPT fluorescence in the nucleus. In HepG2 hepatocytes, 6% of cells had higher GFP-NAMPT fluorescence in the cytoplasm, and 84% had higher GFP-NAMPT fluorescence in the nucleus. In both 3T3-L1 and HepG2 cells, GFP-NAMPT was excluded from the nucleus immediately after mitosis and migrated back into it as the cell cycle progressed. In HepG2 cells, endogenous, untagged NAMPT displayed similar changes with the cell cycle, and in nonmitotic cells, GFP-NAMPT accumulated in the nucleus. Similarly, genotoxic, oxidative, or dicarbonyl stress also caused nuclear NAMPT localization. These interventions also increased poly(ADP-ribosyl) polymerase and sirtuin activity, suggesting an increased cellular demand for NAD. We identified a nuclear localization signal in NAMPT and amino acid substitution in this sequence (424RSKK to ASGA), which did not affect its enzymatic activity, blocked nuclear NAMPT transport, slowed cell growth, and increased histone H3 acetylation. These results suggest that NAMPT is transported into the nucleus where it presumably increases NAD synthesis required for cell proliferation. We conclude that specific inhibition of NAMPT transport into the nucleus might be a potential avenue for managing cancer.

• Keywords
• GFP fusion, NAMPT, cancer, epigenetics, nicotinamide adenine dinucleotide (NAD), nuclear localization, pre–B cell colony enhancing factor (PBEF), sirtuin, visfatin,
• MeSH
• Acrylamides pharmacology   MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus metabolism   MeSH
• 3T3-L1 Cells MeSH
• Hep G2 Cells MeSH
• Cytoplasm metabolism   MeSH
• Histones metabolism   MeSH
• Cell Cycle Checkpoints MeSH
• Humans MeSH
• Mutagenesis, Site-Directed MeSH
• Mice MeSH
• NAD metabolism   MeSH
• Nicotinamide Phosphoribosyltransferase chemistry   genetics   metabolism   MeSH
• Oxidative Stress MeSH
• Piperidines pharmacology   MeSH
• Poly(ADP-ribose) Polymerases metabolism   MeSH
• Cell Proliferation MeSH
• Recombinant Fusion Proteins chemistry   genetics   metabolism   MeSH
• Sirtuins metabolism   MeSH
• Cell Survival drug effects   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Acrylamides MeSH
• Histones MeSH
• N-(4-(1-benzoylpiperidin-4-yl)butyl)-3-(pyridin-3-yl)acrylamide MeSH Browser
• NAD MeSH
• Nicotinamide Phosphoribosyltransferase MeSH
• Piperidines MeSH
• Poly(ADP-ribose) Polymerases MeSH
• Recombinant Fusion Proteins MeSH
• Sirtuins MeSH

Poly(ADP-ribosyl)ation is a reversible post-translational modification synthetized by ADP-ribose transferases and removed by poly(ADP-ribose) glycohydrolase (PARG), which plays important roles in DNA damage repair. While well-studied in somatic tissues, much less is known about poly(ADP-ribosyl)ation in the germline, where DNA double-strand breaks are introduced by a regulated program and repaired by crossover recombination to establish a tether between homologous chromosomes. The interaction between the parental chromosomes is facilitated by meiotic specific adaptation of the chromosome axes and cohesins, and reinforced by the synaptonemal complex. Here, we uncover an unexpected role for PARG in coordinating the induction of meiotic DNA breaks and their homologous recombination-mediated repair in Caenorhabditis elegans. PARG-1/PARG interacts with both axial and central elements of the synaptonemal complex, REC-8/Rec8 and the MRN/X complex. PARG-1 shapes the recombination landscape and reinforces the tightly regulated control of crossover numbers without requiring its catalytic activity. We unravel roles in regulating meiosis, beyond its enzymatic activity in poly(ADP-ribose) catabolism.

• MeSH
• Cell Nucleus metabolism   MeSH
• Caenorhabditis elegans genetics   metabolism   MeSH
• DNA metabolism   MeSH
• DNA Breaks, Double-Stranded * MeSH
• Glycoside Hydrolases genetics   metabolism   MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• DNA Repair physiology   MeSH
• Poly ADP Ribosylation MeSH
• Poly Adenosine Diphosphate Ribose metabolism   MeSH
• Protein Processing, Post-Translational MeSH
• Caenorhabditis elegans Proteins genetics   metabolism   MeSH
• Germ Cells MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• DNA MeSH
• Glycoside Hydrolases MeSH
• Nuclear Proteins MeSH
• poly ADP-ribose glycohydrolase MeSH Browser
• Poly Adenosine Diphosphate Ribose MeSH
• Caenorhabditis elegans Proteins MeSH
• SYP-1 protein, C elegans MeSH Browser

One of the most significant insults that jeopardize cardiomyocyte homeostasis is a surge of reactive oxygen species (ROS) in the failing myocardium. Early growth response factor-1 (Egr-1) has been found to act as a transcriptional regulator in multiple biological processes known to exert deleterious effects on cardiomyocytes. We thus investigated the signaling pathways involved in its regulation by H2O2. Egr-1 mRNA levels were found to be maximally induced after 2 h in H2O2-treated H9c2 cells. Egr-1 respective response at the protein level, was found to be maximally induced after 2 h of treatment with 200 microM H2O2, remaining elevated for 6 h, and declining thereafter. H2O2-induced upregulation of Egr-1 mRNA and protein levels was ablated in the presence of agents inhibiting ERKs pathway (PD98059) and JNKs (SP600125, AS601245). Immunofluorescent experiments revealed H2O2-induced Egr-1 nuclear sequestration to be also ERK- and JNK-dependent. Overall, our results show for the first time the fundamental role of ERKs and JNKs in regulating Egr-1 response to H2O2 treatment in cardiac cells at multiple levels: mRNA, protein and subcellular distribution. Nevertheless, further studies are required to elucidate the specific physiological role of Egr-1 regarding the modulation of gene expression and determination of cell fate.

• MeSH
• Active Transport, Cell Nucleus MeSH
• Cell Nucleus drug effects   enzymology   MeSH
• Cell Line MeSH
• Time Factors MeSH
• Extracellular Signal-Regulated MAP Kinases antagonists & inhibitors   metabolism   MeSH
• Fluorescent Antibody Technique MeSH
• Protein Kinase Inhibitors pharmacology   MeSH
• JNK Mitogen-Activated Protein Kinases antagonists & inhibitors   metabolism   MeSH
• Myocytes, Cardiac drug effects   enzymology   MeSH
• Rats MeSH
• RNA, Messenger metabolism   MeSH
• Hydrogen Peroxide pharmacology   MeSH
• Early Growth Response Protein 1 genetics   metabolism   MeSH
• Up-Regulation MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Egr1 protein, rat MeSH Browser
• Extracellular Signal-Regulated MAP Kinases MeSH
• Protein Kinase Inhibitors MeSH
• JNK Mitogen-Activated Protein Kinases MeSH
• RNA, Messenger MeSH
• Hydrogen Peroxide MeSH
• Early Growth Response Protein 1 MeSH

In order to obtain an active and stable oxidation reactor for daily use in biochemical laboratory we decided to immobilize galactose oxidase orientedly through a carbohydrate chain to the magnetic carriers. We used hydrazide derivatives of non-magnetic and magnetic bead cellulose and of magnetic and non-magnetic poly(HEMA-co-EDMA) microspheres. Activation of the enzyme molecules was done by sodium periodate in the presence of supplements (fucose, CuSO4, catalase). Orientedly immobilized galactose oxidase presents high storage stability and lower susceptibility to inappropriate microenvironmental conditions. Reactor reactivated by three pulses of D-galactose retained practically 100% of its native activity after 6 months. The positive properties of both magnetic carriers were entirely confirmed.

• MeSH
• Cellulose chemistry   MeSH
• Enzymes, Immobilized chemistry   MeSH
• Galactose Oxidase chemistry   MeSH
• Immunoglobulin G chemistry   MeSH
• Periodic Acid chemistry   MeSH
• Magnetics MeSH
• Methacrylates chemistry   MeSH
• Microspheres MeSH
• Nuclear Magnetic Resonance, Biomolecular MeSH
• Polyhydroxyethyl Methacrylate chemistry   MeSH
• Swine MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cellulose MeSH
• Enzymes, Immobilized MeSH
• ethylene dimethacrylate MeSH Browser
• Galactose Oxidase MeSH
• Immunoglobulin G MeSH
• Periodic Acid MeSH
• metaperiodate MeSH Browser
• Methacrylates MeSH
• Polyhydroxyethyl Methacrylate MeSH

Although it is well known that chromosomes are non-randomly organized during interphase, it is not completely clear whether higher-order chromatin structure is transmitted from mother to daughter cells. Therefore, we addressed the question of how chromatin is rearranged during interphase and whether heterochromatin pattern is transmitted after mitosis. We additionally tested the similarity of chromatin arrangement in sister interphase nuclei. We noticed a very active cell rotation during interphase, especially when histone hyperacetylation was induced or transcription was inhibited. This natural phenomenon can influence the analysis of nuclear arrangement. Using photoconversion of Dendra2-tagged core histone H4 we showed that the distribution of chromatin in daughter interphase nuclei differed from that in mother cells. Similarly, the nuclear distribution of heterochromatin protein 1β (HP1β) was not completely identical in mother and daughter cells. However, identity between mother and daughter cells was in many cases evidenced by nucleolar composition. Moreover, morphology of nucleoli, HP1β protein, Cajal bodies, chromosome territories, and gene transcripts were identical in sister cell nuclei. We conclude that the arrangement of interphase chromatin is not transmitted through mitosis, but the nuclear pattern is identical in naturally synchronized sister cells. It is also necessary to take into account the possibility that cell rotation and the degree of chromatin condensation during functionally specific cell cycle phases might influence our view of nuclear architecture.

• MeSH
• Cell Nucleolus drug effects   genetics   ultrastructure   MeSH
• Cell Line MeSH
• Coiled Bodies drug effects   genetics   ultrastructure   MeSH
• Chromosomal Proteins, Non-Histone genetics   metabolism   MeSH
• Dactinomycin pharmacology   MeSH
• Fluorescent Dyes MeSH
• Microscopy, Fluorescence MeSH
• Photochemical Processes MeSH
• Heterochromatin drug effects   genetics   ultrastructure   MeSH
• Histones genetics   metabolism   MeSH
• Chromobox Protein Homolog 5 MeSH
• Histone Deacetylase Inhibitors pharmacology   MeSH
• Protein Synthesis Inhibitors pharmacology   MeSH
• Interphase drug effects   genetics   MeSH
• Hydroxamic Acids pharmacology   MeSH
• Humans MeSH
• RNA, Messenger biosynthesis   MeSH
• Mitosis drug effects   genetics   MeSH
• Mice MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CBX1 protein, human MeSH Browser
• Cbx1 protein, mouse MeSH Browser
• Chromosomal Proteins, Non-Histone MeSH
• Dactinomycin MeSH
• Fluorescent Dyes MeSH
• Heterochromatin MeSH
• Histones MeSH
• Chromobox Protein Homolog 5 MeSH
• Histone Deacetylase Inhibitors MeSH
• Protein Synthesis Inhibitors MeSH
• Hydroxamic Acids MeSH
• RNA, Messenger MeSH
• trichostatin A MeSH Browser

BACKGROUND: Tumor necrosis factor-alpha (TNF-alpha) is known for its selective cytotoxic activity on tumour cells. We analysed the response of HT-29 human colon carcinoma cells to this cytokine. MATERIALS AND METHODS: After TNF-alpha treatment, cell proliferation, cell cycle, reactive oxygen species (ROS) production (flow cytometry), the amount of apoptotic cells (flow cytometry, fluorescence microscopy), cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 activity (Western blotting) were detected. RESULTS: TNF-alpha induced a decrease of cell growth and viability, an accumulation of cells in the S-phase of the cell cycle, an increase of subdiploid cell population and nuclear chromatin condensation and fragmentation, but not sooner than 96-120 hours. However, earlier events characteristic of apoptosis occurred, such as caspase-3 activation, PARP cleavage to 89 kDa fragment and changes in ROS production. CONCLUSION: We demonstrated that, in addition to being an early marker of apoptosis, activation of caspase-3 and degradation of PARP may play a causative role in HT-29 cell death induced by TNF-alpha.

• MeSH
• Apoptosis drug effects   MeSH
• Cell Death drug effects   MeSH
• Cell Division drug effects   MeSH
• HT29 Cells drug effects   enzymology   pathology   MeSH
• Caspase 3 MeSH
• Caspases metabolism   MeSH
• Kinetics MeSH
• Humans MeSH
• Poly(ADP-ribose) Polymerases metabolism   MeSH
• Reactive Oxygen Species metabolism   MeSH
• Tumor Necrosis Factor-alpha pharmacology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• CASP3 protein, human MeSH Browser
• Caspase 3 MeSH
• Caspases MeSH
• Poly(ADP-ribose) Polymerases MeSH
• Reactive Oxygen Species MeSH
• Tumor Necrosis Factor-alpha MeSH

In the present study, the spatial organization of intron-containing pre-mRNAs of Epstein-Barr virus (EBV) genes relative to location of splicing factors is investigated. The intranuclear position of transcriptionally active EBV genes, as well as of nascent transcripts, is found to be random with respect to the speckled accumulations of splicing factors (SC35 domains) in Namalwa cells, arguing against the concept of the locus-specific organization of mRNA genes with respect to the speckles. Microclusters of splicing factors are, however, frequently superimposed on nascent transcript sites. The transcript environment is a dynamic structure consisting of both nascent and released transcripts, i.e., the track-like transcript environment. Both EBV sequences of the chromosome 1 homologue are usually associated with the track, are transcriptionally active, and exhibit in most cases a polar orientation. In contrast to nascent transcripts (in the form of spots), the association of a post-transcriptional pool of viral pre-mRNA (in the form of tracks) with speckles is not random and is further enhanced in transcriptionally silent cells when splicing factors are sequestered in enlarged accumulations. The transcript environment reflects the intranuclear transport of RNA from the sites of transcription to SC35 domains, as shown by concomitant mapping of DNA, RNA, and splicing factors. No clear vectorial intranuclear trafficking of transcripts from the site of synthesis toward the nuclear envelope for export into the cytoplasm is observed. Using Namalwa and Raji cell lines, a correlation between the level of viral gene transcription and splicing factor accumulation within the viral transcript environment has been observed. This supports a concept that the level of transcription can alter the spatial relationship among intron-containing genes, their transcripts, and speckles attributable to various levels of splicing factors recruited from splicing factor reservoirs. Electron microscopic in situ hybridization studies reveal that the released transcripts are directed toward reservoirs of splicing factors organized in clusters of interchromatin granules. Our results point to the bidirectional intranuclear movement of macromolecular complexes between intron-containing genes and splicing factor reservoirs: the recruitment of splicing factors to transcription sites and movement of released transcripts from DNA loci to reservoirs of splicing factors.

• MeSH
• Biological Transport MeSH
• Cell Nucleus genetics   metabolism   ultrastructure   virology   MeSH
• DNA-Directed RNA Polymerases antagonists & inhibitors   metabolism   MeSH
• DNA, Viral genetics   metabolism   MeSH
• Microscopy, Electron MeSH
• Microscopy, Fluorescence MeSH
• Transcription, Genetic genetics   MeSH
• Genome, Viral MeSH
• Heterogeneous-Nuclear Ribonucleoproteins MeSH
• Introns genetics   MeSH
• Nuclear Proteins metabolism   MeSH
• Microscopy, Confocal MeSH
• Humans MeSH
• RNA, Messenger genetics   metabolism   MeSH
• Tumor Cells, Cultured MeSH
• Plasmids genetics   MeSH
• RNA Precursors genetics   metabolism   MeSH
• Ribonucleoproteins metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• Serine-Arginine Splicing Factors MeSH
• Spliceosomes genetics   metabolism   ultrastructure   MeSH
• Genes, Viral genetics   MeSH
• Herpesvirus 4, Human genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA-Directed RNA Polymerases MeSH
• DNA, Viral MeSH
• Heterogeneous-Nuclear Ribonucleoproteins MeSH
• Nuclear Proteins MeSH
• RNA, Messenger MeSH
• RNA Precursors MeSH
• Ribonucleoproteins MeSH
• RNA, Viral MeSH
• Serine-Arginine Splicing Factors MeSH
• SRSF2 protein, human MeSH Browser

BACKGROUND: Serum transferrin levels represent an independent predictor of mortality in patients with liver failure. Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of hepatocyte functions. The aim of this study was to explore whether serum transferrin reflects HNF4α activity. METHODS: Factors regulating transferrin expression in alcoholic hepatitis (AH) were assessed via transcriptomic/methylomic analysis as well as chromatin immunoprecipitation coupled to DNA sequencing. The findings were corroborated in primary hepatocytes. Serum and liver samples from 40 patients with advanced liver disease of multiple etiologies were also studied. RESULTS: In patients with advanced liver disease, serum transferrin levels correlated with hepatic transferrin expression (r = 0.51, p = 0.01). Immunohistochemical and biochemical tests confirmed reduced HNF4α and transferrin protein levels in individuals with cirrhosis. In AH, hepatic gene-gene correlation analysis in liver transcriptome revealed an enrichment of HNF4α signature in transferrin-correlated transcriptome while transforming growth factor beta 1 (TGFβ1), tumor necrosis factor α (TNFα), interleukin 1 beta (IL-1β), and interleukin 6 (IL-6) negatively associated with transferrin signature. A key regulatory region in transferrin promoter was hypermethylated in patients with AH. In primary hepatocytes, treatment with TGFβ1 or the HNF4α inhibitor BI6015 suppressed transferrin production, while exposure to TNFα, IL-1β, and IL-6 had no effect. The correlation between hepatic HNF4A and transferrin mRNA levels was also seen in advanced liver disease. CONCLUSIONS: Serum transferrin levels constitute a prognostic and mechanistic biomarker. Consequently, they may serve as a surrogate of impaired hepatic HNF4α signaling and liver failure.

• Keywords
• Alcoholic hepatitis, Cirrhosis, End-stage liver disease, HNF4alpha, Transferrin,
• MeSH
• Hepatocyte Nuclear Factors metabolism   MeSH
• Hepatocytes metabolism   pathology   MeSH
• Liver Cirrhosis metabolism   MeSH
• Middle Aged MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• DNA Methylation MeSH
• Liver Neoplasms metabolism   MeSH
• Liver Diseases metabolism   pathology   MeSH
• Promoter Regions, Genetic MeSH
• Aged MeSH
• Gene Expression Profiling MeSH
• Transforming Growth Factor beta1 metabolism   MeSH
• Check Tag
• Middle Aged MeSH
• Humans MeSH
• Male MeSH
• Aged MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• Hepatocyte Nuclear Factors MeSH
• RNA, Messenger MeSH
• TGFB1 protein, human MeSH Browser
• Transforming Growth Factor beta1 MeSH

Eukaryotic RNA can carry more than 100 different types of chemical modifications. Early studies have been focused on modifications of highly abundant RNA, such as ribosomal RNA and transfer RNA, but recent technical advances have made it possible to also study messenger RNA (mRNA). Subsequently, mRNA modifications, namely methylation, have emerged as key players in eukaryotic gene expression regulation. The most abundant and widely studied internal mRNA modification is N6 -methyladenosine (m6 A), but the list of mRNA chemical modifications continues to grow as fast as interest in this field. Over the past decade, transcriptome-wide studies combined with advanced biochemistry and the discovery of methylation writers, readers, and erasers revealed roles for mRNA methylation in the regulation of nearly every aspect of the mRNA life cycle and in diverse cellular, developmental, and disease processes. Although large parts of mRNA function are linked to its cytoplasmic stability and regulation of its translation, a number of studies have begun to provide evidence for methylation-regulated nuclear processes. In this review, we summarize the recent advances in RNA methylation research and highlight how these new findings have contributed to our understanding of methylation-dependent RNA processing in the nucleus. This article is categorized under: RNA Processing > RNA Editing and Modification RNA Processing > Splicing Regulation/Alternative Splicing RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

• Keywords
• RNA demethylase, RNA methylase, RNA processing,
• MeSH
• Cell Nucleus metabolism   MeSH
• Epigenesis, Genetic MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Methylation MeSH
• RNA Precursors metabolism   MeSH
• Transcriptome MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH
• RNA Precursors MeSH