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Epigenetic Silencing of Nucleolar rRNA Genes in Alzheimer's Disease  [PDF]
Maciej Pietrzak,Grzegorz Rempala,Peter T. Nelson,Jing-Juan Zheng,Michal Hetman
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0022585
Abstract: Ribosomal deficits are documented in mild cognitive impairment (MCI), which often represents an early stage Alzheimer's disease (AD), as well as in advanced AD. The nucleolar rRNA genes (rDNA), transcription of which is critical for ribosomal biogenesis, are regulated by epigenetic silencing including promoter CpG methylation.
Nucleolar Association and Transcriptional Inhibition through 5S rDNA in Mammals  [PDF]
Andrew M. Fedoriw,Joshua Starmer,Della Yee,Terry Magnuson
PLOS Genetics , 2012, DOI: 10.1371/journal.pgen.1002468
Abstract: Changes in the spatial positioning of genes within the mammalian nucleus have been associated with transcriptional differences and thus have been hypothesized as a mode of regulation. In particular, the localization of genes to the nuclear and nucleolar peripheries is associated with transcriptional repression. However, the mechanistic basis, including the pertinent cis- elements, for such associations remains largely unknown. Here, we provide evidence that demonstrates a 119 bp 5S rDNA can influence nucleolar association in mammals. We found that integration of transgenes with 5S rDNA significantly increases the association of the host region with the nucleolus, and their degree of association correlates strongly with repression of a linked reporter gene. We further show that this mechanism may be functional in endogenous contexts: pseudogenes derived from 5S rDNA show biased conservation of their internal transcription factor binding sites and, in some cases, are frequently associated with the nucleolus. These results demonstrate that 5S rDNA sequence can significantly contribute to the positioning of a locus and suggest a novel, endogenous mechanism for nuclear organization in mammals.
Expression of 5 S rRNA genes linked to 35 S rDNA in plants, their epigenetic modification and regulatory element divergence
Sònia Garcia, Lucie Crhák Khaitová, Ale? Kova?ík
BMC Plant Biology , 2012, DOI: 10.1186/1471-2229-12-95
Abstract: We found that homogenization of L-type units went to completion in most (4/6) but not all species. Two species contained major L-type and minor S-type units (termed Ls-type). The linked genes dominate 5?S rDNA expression while the separate tandems do not seem to be expressed. Members of tribe Anthemideae evolved functional variants of the polymerase III promoter in which a residing C-box element differs from the canonical angiosperm motif by as much as 30%. On this basis, a more relaxed consensus sequence of a plant C-box: (5’-RGSWTGGGTG-3’) is proposed. The 5?S paralogs display heavy DNA methylation similarly as to their unlinked counterparts. FISH revealed the close association of 35?S-5?S arrays with nucleolar periphery indicating that transcription of 5?S genes may occur in this territory.We show that the unusual linked arrangement of 5?S genes, occurring in several plant species, is fully compatible with their expression and functionality. This extraordinary 5?S gene dynamics is manifested at different levels, such as variation in intrachromosomal positions, unit structure, epigenetic modification and considerable divergence of regulatory motifs.Nuclear ribosomal DNA (rDNA) encoding 5?S, 5.8?S, 18?S and 26?S rRNA belong to the most important housekeeping genes playing a central role in cell metabolism [1]. In plant genomes there may be from several hundred up to tens of thousands of highly homogeneous copies of each gene. A high copy number of these genes is probably important to ensure increased demand for proteosynthesis during plant development [2] but other functions, such as stabilization of the cell nucleus, have also been proposed [3]. Each large 35?S (45?S in animals) rDNA unit contains 18?S, 5.8?S and 26?S rRNA genes, the internal transcribed spacers (ITSs), and an intergenic spacer (IGS) (for review see [4]). The 35?S units are organized in tandem arrays at one or several loci. The 5?S rDNA encoding a 120-bp-long transcript has been traditionally cons
SGNP: An Essential Stress Granule/Nucleolar Protein Potentially Involved in 5.8s rRNA Processing/Transport  [PDF]
Chun-Hong Zhu, Jinyong Kim, Jerry W. Shay, Woodring E. Wright
PLOS ONE , 2008, DOI: 10.1371/journal.pone.0003716
Abstract: Background Stress Granules (SG) are sites of accumulation of stalled initiation complexes that are induced following a variety of cellular insults. In a genetic screen for factors involved in protecting human myoblasts from acute oxidative stress, we identified a gene encoding a protein we designate SGNP (Stress Granule and Nucleolar Protein). Methodology/Principal Findings A gene-trap insertional mutagenesis screen produced one insertion that conferred resistance to sodium arsenite. RT-PCR/3′ RACE was used to identify the endogenous gene expressed as a GFP-fusion transcript. SGNP is localized in both the cytoplasm and nucleolus and defines a non-nucleolar compartment containing 5.8S rRNA, a component of the 60S ribosomal subunit. Under oxidative stress, SGNP nucleolar localization decreases and it rapidly co-localizes with stress granules. The decrease in nucleolar SGNP following oxidative stress was accompanied by a large increase in nucleolar 5.8S rRNA. Knockdown of SGNP with shRNA increased global mRNA translation but induced growth arrest and cell death. Conclusions These results suggest that SGNP is an essential gene that may be involved in ribosomal biogenesis and translational control in response to oxidative stress.
Analysis of nucleolar pre-rRNA processing sites in pea (Pisum sativum)
Hong Long,Xianlu Zeng,Mingda Jiao,Bo Hu,Haijing Sun,Zhenlan Liu,Liyong Zhang,Shui Hao
Science China Life Sciences , 2003, DOI: 10.1007/BF03182685
Abstract: The location of rRNA processing was analyzed by usingin situ hybridization with ITS1 probe and immunolabeling of anti-fibrillarin mAb in pea (Pisum sativum) root pole cells. The results showed that rRNA processing sites were in dense fibrillar components (DFCs) and granular components (GCs), but not in fibrillar centers (FCs). Low doses of actinomycin D (AMD) treatment can selectively suppress pre-rRNA synthesis but cannot disturb the processing of preformed pre-rRNAs. With AMD treatment prolonged, the density of labeled signals gradually decreased, indicating the preformed pre-rRNAs were gradually processed.
Nucleolar proteins change in altered gravity
Margarita A. Sobol,Fernando Gonzalez-Camacho,Elizabeth L. Kordyum,Francisco Javier Medina
Journal of Applied Biomedicine , 2007,
Abstract: Nucleolin is a major highly phosphorylated nucleolar protein involved in the regulation of r-chromatincondensation/expansion and rDNA transcription as well as in rRNA processing. The nucleolar proteinhomologous to the mammalian nucleolin and to the onion nucleolin-like protein NopA100, was detectedin the nuclear soluble protein fraction, and in the nuclear matrix fraction from Lepidium sativum rootmeristematic cells, using the selective silver staining method and the cross-reaction with the anti-NopA100 antibody. In 2-DE Western blots of both nuclear fractions, the nucleolin-like protein wasrevealed as a smear on the level of 90 kDa extending through a certain range of pI. In both extractsobtained from seedlings germinated and grown under slow clinorotation, the extension of the pI rangewas shorter and the molecular weight range was thinner than in the 1 g control; moreover, in the nuclearmatrix fraction, the spread of the pI range was separated into two clusters. The results obtained couldindicate a lower phosphorylation of the protein, suggesting a decrease in the activity of L. sativumnucleolin-like protein under clinorotation.
Regulation of Ribosomal RNA Production by RNA Polymerase I: Does Elongation Come First?  [PDF]
Benjamin Albert,Jorge Perez-Fernandez,Isabelle Léger-Silvestre,Olivier Gadal
Genetics Research International , 2012, DOI: 10.1155/2012/276948
Abstract: Ribosomal RNA (rRNA) production represents the most active transcription in the cell. Synthesis of the large rRNA precursors (35–47S) can be achieved by up to 150 RNA polymerase I (Pol I) enzymes simultaneously transcribing each rRNA gene. In this paper, we present recent advances made in understanding the regulatory mechanisms that control elongation. Built-in Pol I elongation factors, such as Rpa34/Rpa49 in budding yeast and PAF53/CAST in humans, are instrumental to the extremely high rate of rRNA production per gene. rRNA elongation mechanisms are intrinsically linked to chromatin structure and to the higher-order organization of the rRNA genes (rDNA). Factors such as Hmo1 in yeast and UBF1 in humans are key players in rDNA chromatin structure in vivo. Finally, elongation factors known to regulate messengers RNA production by RNA polymerase II are also involved in rRNA production and work cooperatively with Rpa49 in vivo. 1. Introduction In cell nuclei, three RNA polymerases transcribe the genome. The most importance is placed on RNA polymerase II (Pol II), which is responsible for synthesizing mRNA and a large variety of noncoding RNAs. The vast majority of RNA production in growing cells is carried out by RNA polymerase I (Pol I), which transcribes the precursor of large rRNA, and by RNA polymerase III (Pol III), which transcribes 5S rRNA, tRNA, and some noncoding RNAs. Observation of cryofixed cryosubstituted other sections analyzed by electron microscopy reveals that exponentially growing budding yeast cells contain up to 104 ribosomes per μm3 [1], which represents up to 10% of the cytoplasmic volume [2] (Figure 1(a)). Figure 1: Budding yeast cells and ribosome production. (a) Morphology of Saccharomyces cerevisiae cells after cryofixation and freeze substitution. Ribosomes are individually localized in the cytoplasm (see individual ribosomes detected in the zoomed region). In the nucleus, the nucleolus (No) is detected as a large electron-dense region compared with low electron density of the nucleoplasm (Np). (b) Morphology of the nucleolus. The nucleus appears outlined by a double envelope with pores, and the nucleolus is in close contact with the nuclear envelope. In the nucleolus, a dense fibrillar network is visible throughout the nucleolar volume. Granular components are dispersed throughout the rest of the nucleolus. (c) Visualization of active genes in rDNA. Using a mutant strain with a reduced number of rDNA copies (strain NOY1071; 25 rDNA copies), Miller spreading of total nucleolar DNA allowed single-gene analysis of rRNA genes. (d)
Characterization of copy numbers of 16S rDNA and 16S rRNA of Candidatus Liberibacter asiaticus and the implication in detection in planta using quantitative PCR
Jeong-soon Kim, Nian Wang
BMC Research Notes , 2009, DOI: 10.1186/1756-0500-2-37
Abstract: We investigated the copy numbers of the 16S rDNA and 16S rRNA of the HLB pathogen and the implication of improving the diagnosis of HLB for early detection using Quantitative PCR. We compared the detection of HLB with different Quantitative PCR based methods with primers/probe targeting either 16S rDNA, beta-operon DNA, 16S rRNA, or beta-operon RNA. The 16S rDNA copy number of Ca. Liberibacter asiaticus was estimated to be three times of that of the beta-operon region, thus allowing detection of lower titer of Ca. L. asiaticus. Quantitative reverse transcriptional PCR (QRT-PCR) indicated that the 16S rRNA averaged 7.83 times more than that of 16S rDNA for the same samples. Dilution analysis also indicates that QRT-PCR targeting 16S rRNA is 10 time more sensitive than QPCR targeting 16S rDNA. Thus QRT-PCR was able to increase the sensitivity of detection by targeting 16S rRNA.Our result indicates that Candidatus Liberibacter asiaticus contains three copies of 16S rDNA. The copy number of 16S rRNA of Ca. L. asiaticus in planta averaged about 7.8 times of 16S rDNA for the same set of samples tested in this study. Detection sensitivity of HLB could be improved through the following approaches: using 16S rDNA based primers/probe in the QPCR assays; and using QRT-PCR assays targeting 16S rRNA.Citrus Huanglongbing (HLB) is one of the most devastating diseases on citrus and is associated with a phloem limited bacterium which has yet to be cultured in vitro. Consequently, the pathogen was given a provisional Candidatus status in nomenclature [1,2]. Currently, three species of the pathogen are recognized from trees with HLB disease based on 16S rDNA sequence: Candidatus Liberibacter asiaticus (Las), Ca. Liberibacter africanus (Laf), and Ca. Liberibacter americanus (Lam); Las is the most prevalent species among HLB infected trees [1,3-5]. Las has been spreading worldwide over the last century and has been identified in Japan, China, Southeast Asia, India, Arabian Peninsula, Br
The Human Nucleolar Protein FTSJ3 Associates with NIP7 and Functions in Pre-rRNA Processing  [PDF]
Luis G. Morello, Patricia P. Coltri, Alexandre J. C. Quaresma, Fernando M. Simabuco, Tereza C. L. Silva, Guramrit Singh, Jeffrey A. Nickerson, Carla C. Oliveira, Melissa J. Moore, Nilson I. T. Zanchin
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0029174
Abstract: NIP7 is one of the many trans-acting factors required for eukaryotic ribosome biogenesis, which interacts with nascent pre-ribosomal particles and dissociates as they complete maturation and are exported to the cytoplasm. By using conditional knockdown, we have shown previously that yeast Nip7p is required primarily for 60S subunit synthesis while human NIP7 is involved in the biogenesis of 40S subunit. This raised the possibility that human NIP7 interacts with a different set of proteins as compared to the yeast protein. By using the yeast two-hybrid system we identified FTSJ3, a putative ortholog of yeast Spb1p, as a human NIP7-interacting protein. A functional association between NIP7 and FTSJ3 is further supported by colocalization and coimmunoprecipitation analyses. Conditional knockdown revealed that depletion of FTSJ3 affects cell proliferation and causes pre-rRNA processing defects. The major pre-rRNA processing defect involves accumulation of the 34S pre-rRNA encompassing from site A′ to site 2b. Accumulation of this pre-rRNA indicates that processing of sites A0, 1 and 2 are slower in cells depleted of FTSJ3 and implicates FTSJ3 in the pathway leading to 18S rRNA maturation as observed previously for NIP7. The results presented in this work indicate a close functional interaction between NIP7 and FTSJ3 during pre-rRNA processing and show that FTSJ3 participates in ribosome synthesis in human cells.
GRIM-1, a Novel Growth Suppressor, Inhibits rRNA Maturation by Suppressing Small Nucleolar RNAs  [PDF]
Shreeram C. Nallar, Limei Lin, Varsha Srivastava, Padmaja Gade, Edward R. Hofmann, Hafiz Ahmed, Sekhar P. Reddy, Dhananjaya V. Kalvakolanu
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0024082
Abstract: We have recently isolated novel IFN-inducible gene, Gene associated with Retinoid-Interferon-induced Mortality-1 (GRIM-1), using a genetic technique. Moderate ectopic expression of GRIM-1 caused growth inhibition and sensitized cells to retinoic acid (RA)/IFN-induced cell death while high expression caused apoptosis. GRIM-1 depletion, using RNAi, conferred a growth advantage. Three protein isoforms (1α, 1β and 1γ) with identical C-termini are produced from GRIM-1 mRNA. We show that GRIM-1 isoforms interact with NAF1 and DKC1, two essential proteins required for box H/ACA sno/sca RNP biogenesis and suppresses box H/ACA RNA levels in mammalian cells by delocalizing NAF1. Suppression of these small RNAs manifests as inefficient rRNA maturation and growth suppression. Interestingly, yeast Shq1p also caused growth suppression in mammalian cells. Consistent with its growth-suppressive property, GRIM-1 expression is lost in a number of human primary prostate tumors. Our observations support a recent study that GRIM-1 might act as a co-tumor suppressor in the prostate.
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