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Katherine E Sloan, Ahmed S Warda, Sunny Sharma, Karl-Dieter Entian, Denis L J Lafontaine, Markus T Bohnsack
Ribosomal RNAs are extensively modified during their transcription and subsequent maturation in the nucleolus, nucleus and cytoplasm. RNA modifications, which are installed either by snoRNA-guided or by stand-alone enzymes, generally stabilise the structure of the ribosome. However, they also cluster at functionally important sites of the ribosome, such as the peptidyltransferase centre and the decoding site, where they facilitate efficient and accurate protein synthesis. The recent identification of sites of substoichiometric 2'-O-methylation and pseudouridylation has overturned the notion that all rRNA modifications are constitutively present on ribosomes, highlighting nucleotide modifications as an important source of ribosomal heterogeneity...
December 2, 2016: RNA Biology
Ei Nakajima, Kouhei Shimaji, Takanari Umegawachi, Saki Tomida, Hideki Yoshida, Nana Yoshimoto, Shingo Izawa, Hiroshi Kimura, Masamitsu Yamaguchi
Epigenetic regulation in starvation is important but not fully understood yet. Here we identified the Rpd3 gene, a Drosophila homolog of histone deacetylase 1, as a critical epigenetic regulator for acquiring starvation stress resistance. Immunostaining analyses of Drosophila fat body revealed that the subcellular localization and levels of Rpd3 dynamically changed responding to starvation stress. In response to starvation stress, the level of Rpd3 rapidly increased, and it accumulated in the nucleolus in what appeared to be foci...
2016: PloS One
Uma Karthika Rajarajacholan, Subhash Thalappilly, Karl Riabowol
Epigenetic, transcriptional and signaling processes in the nucleolus regulate rRNA transcription and cell growth. We report here that the tumor suppressor ING1b binds rDNA, regulates rDNA chromatin modifications and affects nucleolar localization of mTOR to modulate rRNA levels. ING1 represses rDNA transcription by recruiting HDAC1 to rDNA loci, increasing its association with the NoRC complex and deacetylating the histone H3K9 and H3K27 marks of active transcription. Loss of ING1 enhances nucleolar localization of phospho-mTOR and its association with Raptor and GβL, even during rapamycin treatment...
November 29, 2016: Nucleic Acids Research
Hideaki Ishikawa, Harunori Yoshikawa, Keiichi Izumikawa, Yutaka Miura, Masato Taoka, Yuko Nobe, Yoshio Yamauchi, Hiroshi Nakayama, Richard J Simpson, Toshiaki Isobe
Ribosome biogenesis occurs successively in the nucleolus, nucleoplasm, and cytoplasm. Maturation of the ribosomal small subunit is completed in the cytoplasm by incorporation of a particular class of ribosomal proteins and final cleavage of 18S-E pre-rRNA (18S-E). Here, we show that poly(A)-specific ribonuclease (PARN) participates in steps leading to 18S-E maturation in human cells. We found PARN as a novel component of the pre-40S particle pulled down with the pre-ribosome factor LTV1 or Bystin. Reverse pull-down analysis revealed that PARN is a constitutive component of the Bystin-associated pre-40S particle...
November 28, 2016: Nucleic Acids Research
Tamas Gaal, Benjamin P Bratton, Patricia Sanchez-Vazquez, Alexander Sliwicki, Kristine Sliwicki, Andrew Vegel, Rachel Pannu, Richard L Gourse
The spatial organization of DNA within the bacterial nucleoid remains unclear. To investigate chromosome organization in Escherichia coli, we examined the relative positions of the ribosomal RNA (rRNA) operons in space. The seven rRNA operons are nearly identical and separated from each other by as much as 180° on the circular genetic map, a distance of ≥2 million base pairs. By inserting binding sites for fluorescent proteins adjacent to the rRNA operons and then examining their positions pairwise in live cells by epifluorescence microscopy, we found that all but rrnC are in close proximity...
October 15, 2016: Genes & Development
Zeineb Farhane, Franck Bonnier, Hugh J Byrne
Raman microspectroscopy is a non-invasive, in vitro analytical tool which is being increasingly explored for its potential in clinical applications and monitoring the uptake, mechanism of action and cellular interaction at a molecular level of chemotherapeutic drugs, ultimately as a potential label-free preclinical screening and companion diagnostic tool. In this study, doxorubicin (DOX), a "gold standard" chemotherapeutic drug, is employed as a model in the in vitro lung cancer cell line A549 in order to demonstrate the potential of Raman microspectroscopy to screen and identify spectroscopic markers of its trafficking and mechanism of action...
November 25, 2016: Analytical and Bioanalytical Chemistry
Kai Yang, Ming Wang, Yuzheng Zhao, Xuxu Sun, Yi Yang, Xie Li, Aiwu Zhou, Huilin Chu, Hu Zhou, Jianrong Xu, Mian Wu, Jie Yang, Jing Yi
The nucleolus has been recently described as a stress sensor. The nucleoplasmic translocation of nucleolar protein nucleophosmin (NPM1) is a hallmark of nucleolar stress; however, the causes of this translocation and its connection to p53 activation are unclear. Using single live-cell imaging and the redox biosensors, we demonstrate that nucleolar oxidation is a general response to various cellular stresses. During nucleolar oxidation, NPM1 undergoes S-glutathionylation on cysteine 275, which triggers the dissociation of NPM1 from nucleolar nucleic acids...
November 25, 2016: Nature Communications
Yana R Musinova, Olga M Lisitsyna, Dmitry V Sorokin, Eugene A Arifulin, Tatiana A Smirnova, Roman A Zinovkin, Daria M Potashnikova, Yegor S Vassetzky, Eugene V Sheval
Nuclear bodies (NBs) are membraneless organelles that play important roles in genome functioning. A specific type of NBs known as interphase prenucleolar bodies (iPNBs) are formed in the nucleoplasm after hypotonic stress from partially disassembled nucleoli. iPNBs are then disassembled, and the nucleoli are reformed simultaneously. Here, we show that diffusion of B23 molecules from iPNBs but not fusion of iPNBs with the nucleoli contributes to the transfer of B23 from iPNBs to the nucleoli. Maturation of pre-rRNAs and the subsequent outflow of mature rRNAs from iPNBs lead to the disassembly of iPNBs...
November 14, 2016: Journal of Cell Science
Zhuo Chen, XinHua Xu
Nucleolin, a multifunctional protein distributed in the nucleolus, participates in many modulations including rDNA transcription, RNA metabolism, and ribosome assembly. Nucleolin is also found in the cytoplasm and on the cell membrane, and surface nucleolin can bind to various ligands to affect many physiological functions. The expression and localization of nucleolin is often abnormal in cancers, as the differential distribution of nucleolin in cancer can influence the carcinogenesis, proliferation, survival, and metastasis of cancer cells, leading to the cancer progression...
December 2016: Saudi Medical Journal
Jochen Baßler, Yasar Luqman Ahmed, Martina Kallas, Markus Kornprobst, Fabiola R Calviño, Marén Gnädig, Matthias Thoms, Gunter Stier, Sherif Ismail, Satyavati Kharde, Nestor Castillo, Sabine Griesel, Sonja Bastuck, Bettina Bradatsch, Emma Thomson, Dirk Flemming, Irmgard Sinning, Ed Hurt
Ribosome biogenesis in eukaryotic cells is a highly dynamic and complex process innately linked to cell proliferation. The assembly of ribosomes is driven by a myriad of biogenesis factors that shape pre-ribosomal particles by processing and folding the ribosomal RNA and incorporating ribosomal proteins. Biochemical approaches allowed the isolation and characterization of pre-ribosomal particles from Saccharomyces cerevisiae, which lead to a spatiotemporal map of biogenesis intermediates along the path from the nucleolus to the cytoplasm...
November 18, 2016: Protein Science: a Publication of the Protein Society
Roberta Galli, Ortrud Uckermann, Achim Temme, Elke Leipnitz, Matthias Meinhardt, Edmund Koch, Gabriele Schackert, Gerald Steiner, Matthias Kirsch
Coherent anti-Stokes Raman scattering (CARS) microscopy is an emerging technique for identification of brain tumors. However, tumor identification by CARS microscopy on bulk samples and in vivo has been so far verified retrospectively on histological sections, which only provide a gross reference for the interpretation of CARS images without matching at cellular level. Therefore, fluorescent labels were exploited for direct assessment of the interpretation of CARS images of solid and infiltrative tumors. Glioblastoma cells expressing green fluorescent protein (GFP) were used for induction of tumors in mice (n = 7)...
March 11, 2016: Journal of Biophotonics
Lauriel F Earley, John M Powers, Kei Adachi, Joshua T Baumgart, Nancy L Meyer, Qing Xie, Michael S Chapman, Hiroyuki Nakai
: Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown AAP to be essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among the AAV serotypes 1 to 12...
November 16, 2016: Journal of Virology
Ryan T Behrens, Mounavya Aligeti, Ginger M Pocock, Christina A Higgins, Nathan M Sherer
: HIV-1's Rev protein forms a homooligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study we used a functional fluorescently-tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found Rev activity to be remarkably tolerant of diverse NES sequences including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores...
November 16, 2016: Journal of Virology
María Teresa Villoria, Facundo Ramos, Encarnación Dueñas, Peter Faull, Pedro Rodríguez Cutillas, Andrés Clemente-Blanco
Cells are constantly threatened by multiple sources of genotoxic stress that cause DNA damage. To maintain genome integrity, cells have developed a coordinated signalling network called DNA damage response (DDR). While multiple kinases have been thoroughly studied during DDR activation, the role of protein dephosphorylation in the damage response remains elusive. Here, we show that the phosphatase Cdc14 is essential to fulfil recombinational DNA repair in budding yeast. After DNA double-strand break (DSB) generation, Cdc14 is transiently released from the nucleolus and activated...
November 16, 2016: EMBO Journal
Christian E Lamm, Myriam Scherer, Nina Reuter, Bushra Amin, Thomas Stamminger, Uwe Sonnewald
Eukaryotic nuclei are subdivided into subnuclear structures. Among the most prominent of these structures are the nucleolus and the PML nuclear bodies (PML-NBs). PML-NBs are spherical multiprotein aggregates of varying size localized in the interchromosomal area. PML-NB formation is dependent on the presence of the promyelocytic leukemia protein (PML) as well as on post-translational modification of core components by covalent attachment of the small ubiquitin-like modifier (SUMO). So far, PML-NBs as well as PML have been described in mammalian cells only, whereas no orthologs are known in the plant kingdom...
November 2016: FEBS Open Bio
Renjie Wang, Alain Kamgoue, Christophe Normand, Isabelle Léger-Silvestre, Thomas Mangeat, Olivier Gadal
How does the spatial organization of the genome depend on the nuclear shape is unknown, mostly because accurate nuclear size and shape measurement is technically challenging. In large cell populations of the yeast Saccharomyces cerevisiae, we assessed the geometry (size and shape) of nuclei in three dimensions (3D) with a resolution of 30 nm. We improved an automated fluorescent localization method by implementing a post-acquisition correction of the spherical microscopic aberration along the Z-axis, to detect the three dimensional (3D) positions of nuclear pore complexes (NPCs) in the nuclear envelope (NE)...
November 9, 2016: Journal of Cell Science
Hongbo Chen, Liqiao Han, Hsiangi Tsai, Zhiwei Wang, Yanping Wu, Yanhong Duo, Wei Cao, Lijun Chen, Zhirong Tan, Ning Xu, Xianzhang Huang, Junhua Zhuang, Laiqiang Huang
PICT-1 is an essential ribosome biogenesis factor whose loss induces p53 accumulation and apoptosis. Here, we show that DNA damage changes PICT-1 localization and decreases PICT-1 protein levels via the proteasome pathway. Two important phosphatidylinositol 3-kinase-like kinases (PIKKs), ataxia-telangiectasia mutated (ATM) and the Ku70 subunit of DNA-dependent protein kinase (DNA-PK), co-localize and interact with PICT-1 in the nucleolus. Computational prediction of phosphorylation sites and detection using an anti-phospho-substrate antibody suggest that PICT-1 might be a substrate of PIKKs...
November 4, 2016: Oncotarget
Julie Huang, Nahid Iglesias, Danesh Moazed
Chromatin immunoprecipitation (ChIP) is a valuable technique for localizing proteins of interest to specific genomic sites and determining the relative abundance of these proteins at these sites. The ChIP method entails chemical cross-linking of proteins to genomic DNA, isolation of protein-DNA conjugates, and purification of DNA from conjugates. Real-time polymerase chain reactions are used to identify and quantify isolated genomic sequences. Here we describe how to localize yeast proteins to gene sequences residing within the nucleolus, i...
2017: Methods in Molecular Biology
Zhiang Guo, Marisa Segal
Mitotic exit is determined by multiple spatial and temporal cues from the spindle poles and the two compartments in a dividing yeast cell-the mother and the bud. These signals are ultimately integrated by the activation of the mitotic exit network (MEN) to promote persistent release of Cdc14 from the nucleolus. Live imaging analysis using fluorescent protein tags is invaluable to dissect this critical decision-making trigger. Here, we present protocols for routine yeast live cell microscopy applicable to this problem...
2017: Methods in Molecular Biology
Ethel Queralt, José-Antonio Rodriguez-Rodriguez
The phosphatase Cdc14 has a pivotal function in the mitotic exit of Saccharomyces cerevisiae. During interphase, Cdc14 remains inactive in the nucleolus bound to the inhibitor Net1. Cdc14 activation occurs in the metaphase to anaphase transition and it is promoted by at least two signaling pathways called FEAR (CdcFourteen Early Anaphase Release) and MEN (Mitotic Exit Network). These two pathways act in parallel and target the phosphorylation of Net1, thus decreasing Net1 affinity for Cdc14. The activity of Cdc14 can be used as a readout to assay functional interactions of different components of the mitotic exit signaling pathways...
2017: Methods in Molecular Biology
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