DOCSLIB.ORG

Localization of Condensin Subunit XCAP-E in Interphase Nucleus, Nucleoid and Nuclear

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Localization of Condensin Subunit XCAP-E in Interphase Nucleus, Nucleoid and Nuclear 1 Localization of condensin subunit XCAP-E in interphase nucleus, nucleoid and nuclear matrix of XL2 cells. Elmira Timirbulatova, Igor Kireev, Vladimir Ju. Polyakov, and Rustem Uzbekov* Division of Electron Microscopy, A.N.Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899, Moscow, Russia. *Author for correspondence: telephone. 007-095-939-55-28; FAX 007-095-939-31-81 e-mail: [email protected] Key words: XCAP-E; nucleolus; condensin; nuclear matrix; Xenopus. Abbreviations: DAPI , 4’, 6 diamidino-2-phenylindole; DNP, deoxyribonucleoprotein; DRB, 5,6-dichloro-1b-d-ribofuranosylbenzimidazole; SMC, structural maintenance of chromosomes; XCAP-E, Xenopus chromosome associated protein E. 2 Abstract The Xenopus XCAP-E protein is a component of condensin complex In the present work we investigate its localization in interphase XL2 cells and nucleoids. We shown, that XCAP-E is localizes in granular and in dense fibrillar component of nucleolus and also in small karyoplasmic structures (termed “SMC bodies”). Extraction by 2M NaCl does not influence XCAP-E distribution in nucleolus and “SMC bodies”. DNAse I treatment of interphase cells permeabilized by Triton X-100 or nucleoids resulted in partial decrease of labeling intensity in the nucleus, whereas RNAse A treatment resulted in practically complete loss of labeling of nucleolus and “SMC bodies” labeling. In mitotic cells, however, 2M NaCl extraction results in an intense staining of the chromosome region although the labeling was visible along the whole length of sister chromatids, with a stronger staining in centromore region. The data are discussed in view of a hypothesis about participation of XCAP-E in processing of ribosomal RNA. 3 Introduction During the last decades considerable progress has been made in the studies of the structural organization of eukaryotic chromosomes, especially at the lower levels of chromosome compactization (nucleosomes, 30-nm chromatin fiber). However, despite many efforts, principles of higher order chromosome structure and underlying molecular mechanisms remain a matter of debate. Recently, considerable interest has been drawn to a specific class of chromosomal proteins - SMC proteins. It was shown that SMC proteins participate in multiple processes of chromosome dymanics, including mitotic chromosomes condensation, sister chromatid cohesion and segregation, dosage compensation and DNA recombinant reparations (Hirano et al., 1997; Losada et al., 1998; Lieb et al., 1998; Stursberg et al., 1999; Strunnikov et al., 1993; Strunnikov et al., 1995; Jessberger et al., 1996). SMC proteins fell into two major sub-families, of which SMC2/SMC4 are believed to be mainly involved in chromosome compactization. The multisubunit complex consisting of SMC2/SMC4 heterodimer together with regulatory proteins was shown to be required for mitotic chromosomes condensation, hence the term condensin (Hirano, 1999). Recently some data emerged suggesting that condensin subunits may have exerted some additional functions during interphase. For example, condensin subunits were found in the nucleolus of interphase HeLa (Cabello et al., 2001) and XL2 cells (Uzbekov et al., 2003; Timirboulatova et al., 2003). Cabello and colleagues (2001) suggested interaction of SMC proteins with perinucleolar chromatin where they would be involved in reorganizations of ribosomal DNA. This hypothesis is supported by data of chromatin immunoprecipitation experiments performed in S. cerevisiae condensins with ribosomal DNA (Freeman et al., 2000). There is also an alternative hypothesis, based on the studies of condensin dynamics upon inhibition of transcription and processing of ribosomal RNA, suggests that condensin subunits might be involved in pre-ribosome assembly (Uzbekov et al., 2003; Timirboulatova 4 et al., 2003). In addition, a subpopulation of condensin proteins hCAP-C and hCAP-E in human cells and XCAP-E in Xenopus laevis cells was detected in small intranuclear structures of unknown origin (Schmiesing et al., 2000; Uzbekov et al., 2003; Timirboulatova et al., 2003). Colocalization of these SMC-positive structures with phosphorylated form of histone Í3 allowed a speculation that nuclear condensins associated with interphase chromosomes participate in re-initiation of mitotic chromatin condensation (Schmiesing et al., 2000). However, some of these structures also displayed co-localization with nucleolar proteins (Timirboulatova et al., 2003), making them similar to Cajal bodies – specialized nuclear structures where assembly of transcription machinery is thought to take place (Gall, 2000). In the present work, we studied ultrastructural localization of condensin subunit XCAP-E in interphase XL2 cells using immunogold technique, and character of its interaction with different structural components of cell nucleus and nuclear matrix. 5 Results Localization of XCAP-E in interphase cells In interphase XL2 cell nuclei usually have from 1 to 4 nucleoli. In each nucleolus usually contains 1 to 3 fibrillar complexes, consisting of fibrillar centers and dense fibrillar component. Fibrillar complexes are localized in the central area or displaced to the periphery of the nucleolus. The remaining volume is occupied by granular component (Fig 1). To study localization of XCAP-E, two principal ways of cells preparation were used: 1) permeabilization in the 0.5 % Triton X-100 in PBS with subsequent fixation in 3 % paraformaldehyde in the same buffer, or 2) fixation in 3 % paraformaldehyde in PBS and the subsequent permeabilization in 0.5 % Triton X-100. Both methods yielded identical results; therefore permeabilized cells were used for immunolocalization of XCAP-E after enzymatic digestion of nucleic acids. In XL2 cells, XCAP-E colocalized in nucleolus with B23 – the marker protein of granular component. Both proteins were localized in the peripheral zone of the nucleolus, forming a continuous sphere or one to three hemispheres circumventing fibrillar complexes (Fig. 2 a-d). Besides nucleolus, in most of the nuclei antibodies to XCAP-E decorated small globular domains in karyoplasm (Fig. 2 a-d). Fig.1 Ultrastructure of nucleolus in XL2 cell. FC –fibrillar center, DFC – dense fibrillar component, GC – granular component. Bar, 0.5 µm. 6 The number of these domains varied from 2 to 5 per nucleus, with the size ranging from 0.5 to 1 mm. Since another extranucleolar B23-positive domain is known to be Cajal bodies (Gall, 2000 and references therein) we performed double labeling of XL2 cells with antibodies to XCAP-E and coilin – the marker protein of Cajal bodies. Surprisingly, no co- localization of these proteins was found (Fig. 2 e-h). Thus, globular domains containing proteins XCAP-E and B23 are not an equivalent of Cajal bodies in these cells, and possibly represent a special type of intranuclear structures. As XCAP-E belongs to the family of SMC proteins (Hirano et al., 1997), here we term these structures SMC-bodies, for short. Fig. 2 Colocalization of XCAP-E with nucleolar protein B23 or coilin in interphase XL2 cells. Cells were grown and fixed as described under MATERIALS AND METHODS and processed for double immunofluorescence staining with anti-XCAP-E affinity-purified polyclonal antibodies and monoclonal antibodies against human B23 (a-d), or with anti-XCAP-E antibodies and monoclonal antibodies against coilin (e-h). In both cases cells were counterstained with DAPI for DNA visualization (a, d, e, h). Triple DAPI/XCAP-E/B23 labeling (d) and DAPI/XCAP-E/coilin labeling (h) are shown for colocalization of the proteins. Both XCAP-E and B23 were localized in nucleolar periphery as continuous sphere or one - three hemispheres circumventing fibrillar complexes. Besides nucleolus, antibodies to XCAP-E and B23 decorated small globular structures in karyoplasm (d, arrows). Coilin was localized in karyoplasm as numerous fine globular structures. However, in these structures colocalization of coilin with XCAP-E was not observed (h). Bar, 10 µm 7 Fig. 3 Immunoelectron microscopic localization of XCAP-E in control interphase XL2 cells and after actinomycin D and DRB treatments. Cells were grown as described in MATERIALS AND METHODS and fixed without any treatment (a) or after 4h incubation with 5 µg/ml actinomycin D (b) or after 4h incubation with 100 µg/ml DRB (c). After fixation cells were labelled with anti-XCAP-E affinity purified polyclonal antibodies and secondary anti-rabbit antibodies, conjugated with 1 nm gold particles, followed by silver enhancement. In control cells (a) high density of particles was observed within the granular component (GC) and dense fibrillar component (DFC) of the nucleolus. In fibrillar centers (FC) the labeling was absent. After actinomycin D treatment (b) nucleoli were segregated into granular and fibrillar compartments. Labeling was found in granular compartment, but not in fibrillar compartment of segregated nucleolus. In fibrillar compartment the labeling is absent. In cells after DRB treatment (c) XCAP-E labeling was localized in granular component (GC) and dense fibrillar component (DFC) of segregated nucleolus, whereas the fibrillar centers were devoid of gold label. Bar, 0.5 µm. In a previous paper we reported ultrastructural localization of some condensin subunits using colloidal gold-labeled antibodies (Uzbekov et al., 2003). This approach proved to be rather ineffective due to restricted accessibility of antigens in such a dense structures as nucleolus. In the present work, improved procedure was used, which includes aldehyde
Load more

Recommended publications
  • PSF and P54nrb/Nono ^ Multi-Functional Nuclear Proteins
    FEBS 26628 FEBS Letters 531 (2002) 109^114 View metadata, citation and similar papers at core.ac.uk brought to you by CORE Minireview provided by Elsevier - Publisher Connector PSF and p54nrb/NonO ^ multi-functional nuclear proteins Yaron Shav-Tal, Dov Ziporià Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel Received 30 May 2002; revised 10 September 2002; accepted 11 September 2002 First published online 7 October 2002 Edited by Takashi Gojobori glutamine-rich N-terminus of PSF might be involved in pro- Abstract Proteins are often referred to in accordance with the activity with which they were ¢rst associated or the organelle in tein^protein interactions [2]. nrb which they were initially identi¢ed. However, a variety of nu- p54 (human) and NonO (mouse) are highly homologous clear factors act in multiple molecular reactions occurring si- to the C-terminus of PSF (Fig. 1) [7,8]. Proteomics have iden- multaneously within the nucleus. This review describes the func- ti¢ed PSF and p54nrb/NonO in the nucleolus [9] and in asso- tions of the nuclear factors PSF (polypyrimidine tract-binding ciation with the nuclear membrane [10]. p54nrb/NonO was protein-associated splicing factor) and p54nrb/NonO. PSF was recently shown to be a component of a novel nuclear domain initially termed a splicing factor due to its association with the termed paraspeckles [11].TheDrosophila homolog of these nrb second step of pre-mRNA splicing while p54 /NonO was proteins is the NONA/BJ6 protein encoded by the no-on-tran- thought to participate in transcriptional regulation.
    [Show full text]
  • Building the Interphase Nucleus: a Study on the Kinetics of 3D Chromosome Formation, Temporal Relation to Active Transcription, and the Role of Nuclear Rnas
    University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2020-07-28 Building the Interphase Nucleus: A study on the kinetics of 3D chromosome formation, temporal relation to active transcription, and the role of nuclear RNAs Kristin N. Abramo University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Bioinformatics Commons, Cell Biology Commons, Computational Biology Commons, Genomics Commons, Laboratory and Basic Science Research Commons, Molecular Biology Commons, Molecular Genetics Commons, and the Systems Biology Commons Repository Citation Abramo KN. (2020). Building the Interphase Nucleus: A study on the kinetics of 3D chromosome formation, temporal relation to active transcription, and the role of nuclear RNAs. GSBS Dissertations and Theses. https://doi.org/10.13028/a9gd-gw44. Retrieved from https://escholarship.umassmed.edu/ gsbs_diss/1099 Creative Commons License This work is licensed under a Creative Commons Attribution-Noncommercial 4.0 License This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. BUILDING THE INTERPHASE NUCLEUS: A STUDY ON THE KINETICS OF 3D CHROMOSOME FORMATION, TEMPORAL RELATION TO ACTIVE TRANSCRIPTION, AND THE ROLE OF NUCLEAR RNAS A Dissertation Presented By KRISTIN N. ABRAMO Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSPOPHY July 28, 2020 Program in Systems Biology, Interdisciplinary Graduate Program BUILDING THE INTERPHASE NUCLEUS: A STUDY ON THE KINETICS OF 3D CHROMOSOME FORMATION, TEMPORAL RELATION TO ACTIVE TRANSCRIPTION, AND THE ROLE OF NUCLEAR RNAS A Dissertation Presented By KRISTIN N.
    [Show full text]
  • Nuclear Matrix
    Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective Pierre Cau, Claire Navarro, Karim Harhouri, Patrice Roll, Sabine Sigaudy, Elise Kaspi, Sophie Perrin, Annachiara de Sandre-Giovannoli, Nicolas Lévy To cite this version: Pierre Cau, Claire Navarro, Karim Harhouri, Patrice Roll, Sabine Sigaudy, et al.. Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective. Seminars in Cell and Developmental Biology, Elsevier, 2014, 29, pp.125-147. 10.1016/j.semcdb.2014.03.021. hal-01646524 HAL Id: hal-01646524 https://hal-amu.archives-ouvertes.fr/hal-01646524 Submitted on 20 Dec 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Review Nuclear matrix, nuclear envelope and premature aging syndromes in a translational research perspective Pierre Cau a,b,c,∗, Claire Navarro a,b,1, Karim Harhouri a,b,1, Patrice Roll a,b,c,1,2, Sabine Sigaudy a,b,d,1,3, Elise Kaspi a,b,c,1,2, Sophie Perrin a,b,1, Annachiara De Sandre-Giovannoli a,b,d,1,3, Nicolas Lévy a,b,d,∗∗ a Aix-Marseille
    [Show full text]
  • Nuclear Domains
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Cold Spring Harbor Laboratory Institutional Repository CELL SCIENCE AT A GLANCE 2891 Nuclear domains dynamic structures and, in addition, nuclear pore complex has been shown to rapid protein exchange occurs between have a remarkable substructure, in which David L. Spector many of the domains and the a basket extends into the nucleoplasm. Cold Spring Harbor Laboratory, One Bungtown nucleoplasm (Misteli, 2001). An The peripheral nuclear lamina lies Road, Cold Spring Harbor, NY 11724, USA extensive effort is currently underway by inside the nuclear envelope and is (e-mail: [email protected]) numerous laboratories to determine the composed of lamins A/C and B and is biological function(s) associated with thought to play a role in regulating Journal of Cell Science 114, 2891-2893 (2001) © The Company of Biologists Ltd each domain. The accompanying poster nuclear envelope structure and presents an overview of commonly anchoring interphase chromatin at the The mammalian cell nucleus is a observed nuclear domains. nuclear periphery. Internal patches of membrane-bound organelle that contains lamin protein are also present in the the machinery essential for gene The nucleus is bounded by a nuclear nucleoplasm (Moir et al., 2000). The expression. Although early studies envelope, a double-membrane structure, cartoon depicts much of the nuclear suggested that little organization exists of which the outer membrane is envelope/peripheral lamina as within this compartment, more contiguous with the rough endoplasmic transparent, so that internal structures contemporary studies have identified an reticulum and is often studded with can be more easily observed.
    [Show full text]
  • Association of DNA with Nuclear Matrix in in Vitro Assembled Nuclei
    Cell Research (1997), 7, 107-117 Association of DNA with nuclear matrix in in vitro as- sembled nuclei induced by rDNA from Tetrahymena shang- haiensis in Xenopus egg extracts CHEN YING, BO ZHANG, XIU FEN LI, ZHONG HE ZHAI Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing 100871 ABSTRACT The nuclei assembled from exogenous DNA or chro- matin in egg extracts resemble their in vivo counterparts in many aspects. However, the distribution pattern of DNA in these nuclei remains unknown. We introduced rDNA from the macronuclei of Tetrahymena into Xenopus cell- free extracts to examine the association of specific DNA sequences with nuclear matrix (NM) in the nuclei assem- bled in vitro. Our previous works showed the 5'NTS (non- transcription sequences) of the rDNA specifically bind to the NM system in the macronuclei. We show now the rDNA could induce chromatin assembly and nuclear for- mation in Xenopus cell-free system. When we extracted the NM system and compared the binding affinity of differ- ent regions of rDNA with the NM system, we found that the 5'NTS still hold their binding affinity with insoluble structure of the assembled nuclei in the extracts of Xeno- pus eggs. Key words: Nuclear assembly, nuclear matrix, Xeno- pus egg extracts, Tetrahymena rDNA. On the occasion of Professor Lu Ji SHI's (L. C. Sze), eightieth birthday, we present this paper and extend our sincere greetings to Professor SHI. As we mentioned in our paper, in early 1950's, it is Professor SHI who first studied the behavior of exogenous homologous desoxyribose nucleoprotein (chromatin) in amphibian eggs.
    [Show full text]
  • Monoclonal Antibody to SMC2 / CAPE - Purified
    OriGene Technologies, Inc. OriGene Technologies GmbH 9620 Medical Center Drive, Ste 200 Schillerstr. 5 Rockville, MD 20850 32052 Herford UNITED STATES GERMANY Phone: +1-888-267-4436 Phone: +49-5221-34606-0 Fax: +1-301-340-8606 Fax: +49-5221-34606-11 [email protected] [email protected] AM05324PU-N Monoclonal Antibody to SMC2 / CAPE - Purified Alternate names: CAP-E, Chromosome-associated protein E, SMC protein 2, SMC-2, SMC2L1, Structural maintenance of chromosomes protein 2, XCAP-E homolog Quantity: 0.1 mg Concentration: 1.0 mg/ml Background: CAP-E and CAP-C (Chromosome Associated Protein-E & C) are also SMC (Structural Maintenance of Chromosome) family members that form a heterodimeric complex required for mitotic chromosome condensation to achieve proper segregation of genetic information during subsequent cell division. hCAP-C/hCAP-E is a component of a multiprotein complex called condensin that interacts with phosphorylation of Histone H3 resulting in the mitotic chromosome condensation. The distribution patterns of the two heterodimeric complexes in interphase nucleus indicate independent behaviour of the two complexes during cell cycle. These results suggest that the two distinct complexes are involved in different aspects of mitotic chromosome organization in human cells. Uniprot ID: O95347 NCBI: NP_001036015.1 GeneID: 10592 Host / Isotype: Mouse / IgG Clone: E1M Immunogen: Hybridoma produced by the fusion of splenocytes from mice immunized with recombinant protein corresponding to amino acids 523-768 of Human CAP-E and Mouse myeloma cells. Genename: SMC2 Format: State: Liquid purified IgG fraction. Buffer System: PBS containing 0.08% Sodium Azide as preservative. Applications: Western Blot: 1-5 µg/ml.
    [Show full text]
  • Supplementary Table S1. Correlation Between the Mutant P53-Interacting Partners and PTTG3P, PTTG1 and PTTG2, Based on Data from Starbase V3.0 Database
    Supplementary Table S1. Correlation between the mutant p53-interacting partners and PTTG3P, PTTG1 and PTTG2, based on data from StarBase v3.0 database. PTTG3P PTTG1 PTTG2 Gene ID Coefficient-R p-value Coefficient-R p-value Coefficient-R p-value NF-YA ENSG00000001167 −0.077 8.59e-2 −0.210 2.09e-6 −0.122 6.23e-3 NF-YB ENSG00000120837 0.176 7.12e-5 0.227 2.82e-7 0.094 3.59e-2 NF-YC ENSG00000066136 0.124 5.45e-3 0.124 5.40e-3 0.051 2.51e-1 Sp1 ENSG00000185591 −0.014 7.50e-1 −0.201 5.82e-6 −0.072 1.07e-1 Ets-1 ENSG00000134954 −0.096 3.14e-2 −0.257 4.83e-9 0.034 4.46e-1 VDR ENSG00000111424 −0.091 4.10e-2 −0.216 1.03e-6 0.014 7.48e-1 SREBP-2 ENSG00000198911 −0.064 1.53e-1 −0.147 9.27e-4 −0.073 1.01e-1 TopBP1 ENSG00000163781 0.067 1.36e-1 0.051 2.57e-1 −0.020 6.57e-1 Pin1 ENSG00000127445 0.250 1.40e-8 0.571 9.56e-45 0.187 2.52e-5 MRE11 ENSG00000020922 0.063 1.56e-1 −0.007 8.81e-1 −0.024 5.93e-1 PML ENSG00000140464 0.072 1.05e-1 0.217 9.36e-7 0.166 1.85e-4 p63 ENSG00000073282 −0.120 7.04e-3 −0.283 1.08e-10 −0.198 7.71e-6 p73 ENSG00000078900 0.104 2.03e-2 0.258 4.67e-9 0.097 3.02e-2 Supplementary Table S2.
    [Show full text]
  • Lorena Novoa-Aponte
    A Strain: fl/fl ∆hep Strain: fl/fl ∆hep AnalysisAAV: ofLuc the ironLuc chaperonePCBP1-WT PCBP1PCBP1-∆Fe interactome:PCBP1-∆RNA PCBP1 variant intersection of DNA repair and iron traffickingAAV: Luc Luc WT ∆Fe ∆RNA PCBP1 −40 Lorena Novoa-Aponte a*, Sarju J. Patel a, Olga Protchenko a, James Wohlschlegel b, Caroline C. Philpott a −40 PCBP1, IHC PCBP1, GAPDH - a Genetics and Metabolism Section,a NIDDK, NIH, Bethesda, MD, USA. b Department of Biological Chemistry, UCLA, Los Angeles, CA, USA C.C. Philpott, et al. *[email protected] 80 B P = 0.0262 60 40 1. Iron is essential but toxic 2. Increased DNA damage in cells lacking PCBP1 ns H&E ? + 20 C.C. Philpott, et al. Iron is used as an essential cofactor by several enzymes involved in DNA ⦿ Increased TUNEL in cells and tissue livers from mice lacking PCBP1. ? replication and repair. However, unchaperoned iron promotes redox ⦿ PCBP1 binds both, iron and single-stranded nucleicTG, nmol/mg protein 0 acids. stress that may affect DNA stability. Strain: Δhep Δhep Δhep ? ⦿ The iron binding activity of PCBP1 controls suppressionAAV: of DNA damage C WT ∆Fe ∆RNA PCBP1 variant ? Iron storage HEK293 cells Mouse Liver ? A 8 siNT+P1var 100 ? A 8 var siPCBP1 2 ? Mammals use the iron siNT+P1 ns var Fe-S cluster assembly 6 siPCBP1+P1siPCBP1 80 ADI1 TUNEL chaperone PCBP1 to var ? 6 siPCBP1+P1 metalate several iron- 60 = 0.0468 = 0.0354 = 0.0465 cell / mm population P P P population + dependent enzymes + 4 + 4 ? 40 Degradation of HIF1α Fig. 3. Iron chaperone-mediated handling of cytosolic labile iron pool.
    [Show full text]
  • A Yeast Phenomic Model for the Influence of Warburg Metabolism on Genetic Buffering of Doxorubicin Sean M
    Santos and Hartman Cancer & Metabolism (2019) 7:9 https://doi.org/10.1186/s40170-019-0201-3 RESEARCH Open Access A yeast phenomic model for the influence of Warburg metabolism on genetic buffering of doxorubicin Sean M. Santos and John L. Hartman IV* Abstract Background: The influence of the Warburg phenomenon on chemotherapy response is unknown. Saccharomyces cerevisiae mimics the Warburg effect, repressing respiration in the presence of adequate glucose. Yeast phenomic experiments were conducted to assess potential influences of Warburg metabolism on gene-drug interaction underlying the cellular response to doxorubicin. Homologous genes from yeast phenomic and cancer pharmacogenomics data were analyzed to infer evolutionary conservation of gene-drug interaction and predict therapeutic relevance. Methods: Cell proliferation phenotypes (CPPs) of the yeast gene knockout/knockdown library were measured by quantitative high-throughput cell array phenotyping (Q-HTCP), treating with escalating doxorubicin concentrations under conditions of respiratory or glycolytic metabolism. Doxorubicin-gene interaction was quantified by departure of CPPs observed for the doxorubicin-treated mutant strain from that expected based on an interaction model. Recursive expectation-maximization clustering (REMc) and Gene Ontology (GO)-based analyses of interactions identified functional biological modules that differentially buffer or promote doxorubicin cytotoxicity with respect to Warburg metabolism. Yeast phenomic and cancer pharmacogenomics data were integrated to predict differential gene expression causally influencing doxorubicin anti-tumor efficacy. Results: Yeast compromised for genes functioning in chromatin organization, and several other cellular processes are more resistant to doxorubicin under glycolytic conditions. Thus, the Warburg transition appears to alleviate requirements for cellular functions that buffer doxorubicin cytotoxicity in a respiratory context.
    [Show full text]
  • Gemin4 Is an Essential Gene in Mice, and Its Overexpression in Human Cells Causes Relocalization of the SMN Complex to the Nucleoplasm Ingo D
    © 2018. Published by The Company of Biologists Ltd | Biology Open (2018) 7, bio032409. doi:10.1242/bio.032409 RESEARCH ARTICLE Gemin4 is an essential gene in mice, and its overexpression in human cells causes relocalization of the SMN complex to the nucleoplasm Ingo D. Meier1,*,§, Michael P. Walker1,2,‡,§ and A. Gregory Matera¶ ABSTRACT nuclear ribonucleoproteins (snRNPs). Each of these snRNPs Gemin4 is a member of the Survival Motor Neuron (SMN) protein contains a common set of seven RNA binding factors, called Sm complex, which is responsible for the assembly and maturation of Sm- proteins, that forms a heptameric ring around the snRNA, known as class small nuclear ribonucleoproteins (snRNPs). In metazoa, Sm the Sm core. Biogenesis of the Sm core is carried out by another snRNPs are assembled in the cytoplasm and subsequently imported macromolecular assemblage called the Survival Motor Neuron into the nucleus. We previously showed that the SMN complex is (SMN) complex, consisting of at least nine proteins (Gemins 2-8, required for snRNP import in vitro, although it remains unclear which unrip and SMN) (reviewed in Battle et al., 2006a; Matera et al., specific components direct this process. Here, we report that Gemin4 2007; Matera and Wang, 2014). overexpression drives SMN and the other Gemin proteins from the Following RNA polymerase II-mediated transcription in the cytoplasm into the nucleus. Moreover, it disrupts the subnuclear nucleus, pre-snRNAs are exported to the cytoplasm for assembly localization of the Cajal body marker protein, coilin, in a dose- into stable RNP particles (Jarmolowski et al., 1994; Ohno et al., dependent manner.
    [Show full text]
  • Nuclear Matrix Protein Matrin 3 Is a Regulator of ZAP-Mediated Retroviral
    Erazo and Goff. Retrovirology (2015) 12:57 DOI 10.1186/s12977-015-0182-4 RESEARCH Open Access Nuclear matrix protein Matrin 3 is a regulator of ZAP‑mediated retroviral restriction Angela Erazo1 and Stephen P Goff1,2* Abstract Background: Matrin 3 is a nuclear matrix protein involved in multiple nuclear processes. In HIV-1 infection, Matrin 3 serves as a Rev cofactor important for the cytoplasmic accumulation of HIV-1 transcripts. ZAP is a potent host restric- tion factor of multiple viruses including retroviruses HIV-1 and MoMuLV. In this study we sought to further character- ize Matrin 3 functions in the regulation of HIV gene expression. Results: Here we describe a function for Matrin 3 as a negative regulator of the ZAP-mediated restriction of retro- viruses. Mass spectrometry analysis of Matrin 3-associated proteins uncovered interactions with proteins of the ZAP degradation complex, DDX17 and EXOSC3. Coimmunoprecipitation studies confirmed Matrin 3 associations with DDX17, EXOSC3 and ZAP, in a largely RNA-dependent manner, indicating that RNA is mediating the Matrin 3 interac- tions with these components of the ZAP degradation complex. Silencing Matrin 3 expression caused a remarkably enhanced ZAP-driven inhibition of HIV-1 and MoMuLV luciferase reporter viruses. This effect was shared with addi- tional nuclear matrix proteins. ZAP targets multiply-spliced HIV-1 transcripts, but in the context of Matrin 3 suppres- sion, this ZAP restriction was broadened to unspliced and multiply-spliced RNAs. Conclusions: Here we reveal an unprecedented role for a nuclear matrix protein, Matrin 3, in the regulation of ZAP’s antiretroviral activity.
    [Show full text]
  • Direct Interaction Between Hnrnp-M and CDC5L/PLRG1 Proteins Affects Alternative Splice Site Choice
    Direct interaction between hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond To cite this version: David Llères, Marco Denegri, Marco Biggiogera, Paul Ajuh, Angus Lamond. Direct interaction be- tween hnRNP-M and CDC5L/PLRG1 proteins affects alternative splice site choice. EMBO Reports, EMBO Press, 2010, 11 (6), pp.445 - 451. 10.1038/embor.2010.64. hal-03027049 HAL Id: hal-03027049 https://hal.archives-ouvertes.fr/hal-03027049 Submitted on 26 Nov 2020 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. scientificscientificreport report Direct interaction between hnRNP-M and CDC5L/ PLRG1 proteins affects alternative splice site choice David Lle`res1*, Marco Denegri1*w,MarcoBiggiogera2,PaulAjuh1z & Angus I. Lamond1+ 1Wellcome Trust Centre for Gene Regulation & Expression, College of Life Sciences, University of Dundee, Dundee, UK, and 2LaboratoriodiBiologiaCellulareandCentrodiStudioperl’IstochimicadelCNR,DipartimentodiBiologiaAnimale, Universita’ di Pavia, Pavia, Italy Heterogeneous nuclear ribonucleoprotein-M (hnRNP-M) is an and affect the fate of heterogeneous nuclear RNAs by influencing their abundant nuclear protein that binds to pre-mRNA and is a structure and/or by facilitating or hindering the interaction of their component of the spliceosome complex.
    [Show full text]