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Endogenous Reverse Transcriptase and Rnase H-Mediated Antiviral Mechanism in Embryonic Stem Cells
www.nature.com/cr www.cell-research.com ARTICLE Endogenous reverse transcriptase and RNase H-mediated antiviral mechanism in embryonic stem cells Junyu Wu1, Chunyan Wu1, Fan Xing1, Liu Cao1, Weijie Zeng1, Liping Guo1, Ping Li1, Yongheng Zhong1, Hualian Jiang1, Manhui Luo1, Guang Shi2, Lang Bu1, Yanxi Ji1, Panpan Hou1, Hong Peng1, Junjiu Huang2, Chunmei Li1 and Deyin Guo 1 Nucleic acid-based systems play important roles in antiviral defense, including CRISPR/Cas that adopts RNA-guided DNA cleavage to prevent DNA phage infection and RNA interference (RNAi) that employs RNA-guided RNA cleavage to defend against RNA virus infection. Here, we report a novel type of nucleic acid-based antiviral system that exists in mouse embryonic stem cells (mESCs), which suppresses RNA virus infection by DNA-mediated RNA cleavage. We found that the viral RNA of encephalomyocarditis virus can be reverse transcribed into complementary DNA (vcDNA) by the reverse transcriptase (RTase) encoded by endogenous retrovirus-like elements in mESCs. The vcDNA is negative-sense single-stranded and forms DNA/RNA hybrid with viral RNA. The viral RNA in the heteroduplex is subsequently destroyed by cellular RNase H1, leading to robust suppression of viral growth. Furthermore, either inhibition of the RTase activity or depletion of endogenous RNase H1 results in the promotion of virus proliferation. Altogether, our results provide intriguing insights into the antiviral mechanism of mESCs and the antiviral function of endogenized retroviruses and cellular RNase H. Such a natural nucleic acid-based antiviral mechanism in mESCs is referred to as ERASE (endogenous RTase/RNase H-mediated antiviral system), which is an addition to the previously known nucleic acid-based antiviral mechanisms including CRISPR/Cas in bacteria and RNAi in plants and invertebrates. -
Specific Principles of Genome-Wide RNA-Chromatin Interactions
ARTICLE There are amendments to this paper https://doi.org/10.1038/s41467-020-14337-6 OPEN RADICL-seq identifies general and cell type–specific principles of genome-wide RNA-chromatin interactions Alessandro Bonetti 1,2,18*, Federico Agostini 3,18, Ana Maria Suzuki1,4, Kosuke Hashimoto1, Giovanni Pascarella1, Juliette Gimenez 5, Leonie Roos6,7, Alex J. Nash6,7, Marco Ghilotti1, Christopher J. F. Cameron8,9, Matthew Valentine 1, Yulia A. Medvedeva10,11,12, Shuhei Noguchi1, Eneritz Agirre 2, Kaori Kashi1, Samudyata2, Joachim Luginbühl1, Riccardo Cazzoli13, Saumya Agrawal1, Nicholas M. Luscombe 3,14,15, Mathieu Blanchette8, Takeya Kasukawa 1, Michiel de Hoon1, Erik Arner1, 1234567890():,; Boris Lenhard 6,7,16, Charles Plessy 1, Gonçalo Castelo-Branco 2, Valerio Orlando5,17* & Piero Carninci 1* Mammalian genomes encode tens of thousands of noncoding RNAs. Most noncoding tran- scripts exhibit nuclear localization and several have been shown to play a role in the reg- ulation of gene expression and chromatin remodeling. To investigate the function of such RNAs, methods to massively map the genomic interacting sites of multiple transcripts have been developed; however, these methods have some limitations. Here, we introduce RNA And DNA Interacting Complexes Ligated and sequenced (RADICL-seq), a technology that maps genome-wide RNA–chromatin interactions in intact nuclei. RADICL-seq is a proximity ligation-based methodology that reduces the bias for nascent transcription, while increasing genomic coverage and unique mapping rate efficiency compared with existing methods. RADICL-seq identifies distinct patterns of genome occupancy for different classes of tran- scripts as well as cell type–specific RNA-chromatin interactions, and highlights the role of transcription in the establishment of chromatin structure. -
Redundancy in Ribonucleotide Excision Repair: Competition, Compensation, and Cooperation
DNA Repair 29 (2015) 74–82 Contents lists available at ScienceDirect DNA Repair j ournal homepage: www.elsevier.com/locate/dnarepair Redundancy in ribonucleotide excision repair: Competition, compensation, and cooperation ∗ Alexandra Vaisman, Roger Woodgate Laboratory of Genomic Integrity, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892-3371, USA a r t i c l e i n f o a b s t r a c t Article history: The survival of all living organisms is determined by their ability to reproduce, which in turn depends Received 1 November 2014 on accurate duplication of chromosomal DNA. In order to ensure the integrity of genome duplication, Received in revised form 7 February 2015 DNA polymerases are equipped with stringent mechanisms by which they select and insert correctly Accepted 9 February 2015 paired nucleotides with a deoxyribose sugar ring. However, this process is never 100% accurate. To fix Available online 16 February 2015 occasional mistakes, cells have evolved highly sophisticated and often redundant mechanisms. A good example is mismatch repair (MMR), which corrects the majority of mispaired bases and which has been Keywords: extensively studied for many years. On the contrary, pathways leading to the replacement of nucleotides Ribonucleotide excision repair with an incorrect sugar that is embedded in chromosomal DNA have only recently attracted significant Nucleotide excision repair attention. This review describes progress made during the last few years in understanding such path- Mismatch repair Ribonuclease H ways in both prokaryotes and eukaryotes. Genetic studies in Escherichia coli and Saccharomyces cerevisiae Flap endonuclease demonstrated that MMR has the capacity to replace errant ribonucleotides, but only when the base is DNA polymerase I mispaired. -
Supporting Information
Supporting Information Figure S1. The functionality of the tagged Arp6 and Swr1 was confirmed by monitoring cell growth and sensitivity to hydeoxyurea (HU). Five-fold serial dilutions of each strain were plated on YPD with or without 50 mM HU and incubated at 30°C or 37°C for 3 days. Figure S2. Localization of Arp6 and Swr1 on chromosome 3. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 3L, Tel 3R, CEN3, and the RP gene are shown under the panels. Figure S3. Localization of Arp6 and Swr1 on chromosome 4. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) in the whole chromosome region are compared. The position of Tel 4L, Tel 4R, CEN4, SWR1, and RP genes are shown under the panels. Figure S4. Localization of Arp6 and Swr1 on the region including the SWR1 gene of chromosome 4. The binding of Arp6- FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position and orientation of the SWR1 gene is shown. Figure S5. Localization of Arp6 and Swr1 on chromosome 5. The binding of Arp6-FLAG (top), Swr1-FLAG (middle), and Arp6-FLAG in swr1 cells (bottom) are compared. The position of Tel 5L, Tel 5R, CEN5, and the RP genes are shown under the panels. Figure S6. Preferential localization of Arp6 and Swr1 in the 5′ end of genes. Vertical bars represent the binding ratio of proteins in each locus. -
Potent Inhibition of Human Telomerase by U-73122
Journal of Biomedical Science (2006) 13:667–674 667 DOI 10.1007/s11373-006-9100-z Potent inhibition of human telomerase by U-73122 Yi-Jui Chen, Wei-Yun Sheng, Pei-Rong Huang & Tzu-Chien V. Wang* Department of Molecular and Cellular Biology, Chang Gung University, Kwei-San, Tao-Yuan, 333, Taiwan Received 28 April 2006; accepted 14 June 2006 Ó 2006 National Science Council, Taipei Key words: alkylating agents, cancer therapyzU-73122, N-ethylmaleimide, telomerase inhibitor Summary Telomerase activity is repressed in normal human somatic cells, but is activated in most cancers, suggesting that telomerase may be an important target for cancer therapy. In this study, we report that U-73122, an amphiphilic alkylating agent that is commonly used as an inhibitor for phospholipase C, is also a potent and selective inhibitor of human telomerase. The inhibition of telomerase by U-73122 was attributed primarily to the pyrrole-2,5-dione group, since its structural analog U-73343 did not inhibit telomerase. In confirmation, we observed that telomerase was inhibited by N-ethylmaleimide, but not N-ethylsuccinimide. The IC50 value of U-73122 for the in vitro inhibition of telomerase activity is 0.2 lM, which is comparable to or slightly more sensitive than that for phospholipase C. The inhibitory action of U-73122 on telomerase appears to be rather selective since the presence of externally added proteins did not protect the inhibition and the IC50 values for the other enzymes tested in this study were at least an order of magnitude higher than that for telomerase. Furthermore, we demonstrate that U-73122 can inhibit telomerase in hemato- poietic cancer cells. -
Pharmacokinetics, Pharmacodynamics and Metabolism Of
PHARMACOKINETICS, PHARMACODYNAMICS AND METABOLISM OF GTI-2040, A PHOSPHOROTHIOATE OLIGONUCLEOTIDE TARGETING R2 SUBUNIT OF RIBONUCLEOTIDE REDUCTASE DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Xiaohui Wei, M.S. * * * * * * The Ohio State University 2006 Approved by Dissertation Committee: Dr. Kenneth K. Chan, Adviser Adviser Dr. Guido Marcucci, Co-adviser Graduate Program in Pharmacy Dr. Thomas D. Schmittgen Dr. Robert J. Lee Co-Adviser Graduate Program in Pharmacy ABSTRACT Over the last several decades, antisense therapy has been developed into a promising gene-targeted strategy to specifically inhibit the gene expression. Ribonucleotide reductase (RNR), composing of subunits R1 and R2, is an important enzyme involved in the synthesis of all of the precursors used in DNA replication. Over- expression of R2 has been found in almost every type of cancer studied. GTI-2040 is a 20-mer phosphorothioate oligonucleotide targeting the coding region in mRNA of the R2 component of human RNR. In this project, clinical pharamcokinetics (PK), pharmacodynamics (PD) and metabolism of this novel therapeutics were investigated in patients with acute myeloid leukemia (AML). A picomolar specific hybridization-ligation ELISA method has been developed and validated for quantification of GTI-2040. GTI-2040 and neophectin complex was found to enhance drug cellular uptake and exhibited sequence- and dose-dependent down-regulation of R2 mRNA and protein in K562 cells. Robust intracellular concentrations (ICs) of GTI-2040 were achieved in peripheral blood mononuclear cells (PBMC) and bone marrow (BM) cells from treated AML patients. GTI-2040 concentrations in the nucleus of BM cells were found to correlate with the R2 mRNA down-regulation and disease response. -
Termin Translat Trna Utr Mutat Protein Signal
Drugs & Chemicals 1: Tumor Suppressor Protein p53 2: Heterogeneous-Nuclear Ribonucleo- (1029) proteins (14) activ apoptosi arf cell express function inactiv induc altern assai associ bind mdm2 mutat p53 p73 pathwai protein regul complex detect exon famili genom respons suppress suppressor tumor wild-typ interact intron isoform nuclear protein sensit site specif splice suggest variant 3: RNA, Transfer (110) 4: DNA Primers (1987) codon contain differ eukaryot gene initi amplifi analysi chain clone detect dna express mrna protein region ribosom rna fragment gene genotyp mutat pcr sequenc site speci suggest synthesi polymorph popul primer reaction region restrict sequenc speci termin translat trna utr 5: Saccharomyces cerevisiae Proteins 6: Apoptosis Regulatory Proteins (291) (733) activ apoptosi apoptosis-induc albican bud candida cerevisia complex encod apoptot bcl-2 caspas caspase-8 cell eukaryot fission function growth interact involv death fasl induc induct ligand methyl necrosi pathwai program sensit surviv trail mutant pomb protein requir saccharomyc strain suggest yeast 7: Plant Proteins (414) 8: Membrane Proteins (1608) access arabidopsi cultivar flower hybrid leaf leav apoptosi cell conserv domain express function gene human identifi inhibitor line maiz plant pollen rice root seed mammalian membran mice mous mutant seedl speci thaliana tomato transgen wheat mutat protein signal suggest transport 1 9: Tumor Suppressor Proteins (815) 10: 1-Phosphatidylinositol 3-Kinase activ arrest cell cycl cyclin damag delet dna (441) 3-kinas activ -
E. Coli Ribonuclease H (Rnase H), Recombinant # 02-060 1,000 Units, # 02-060-5 5 X 1,000 Units
Manufacturer E. coli Ribonuclease H (RNase H), Recombinant # 02-060 1,000 units, # 02-060-5 5 x 1,000 units Ribonuclease H (RNase H) is an endoribonuclease which specifically degrades the RNA strand of an RNA/DNA hybrid, leaving the DNA strand and unhybridized RNA intact. E. coli RNase H (RNaseHI) was over-expressed in E. coli as a recombinant protein and the protein then purified. MW is 17.6 kDa. Applications 1. Removal of mRNA in DNA/RNA hybrid prior to the synthesis of the second strand of cDNA (1, 2) 2. Removal of poly (A) tails from mRNA after hybridization with oligo (dT) (3) 3. Oligodeoxyribonucleotide-directed site-specific cleavage of RNA (4) Specification Form: 50 units/ul in 20mM Tris-HCl (pH 7.5), 100mM KCl, 1mM DTT, 50% Glycerol Specific Activity: 100,000 units/mg protein Unit Definition: 1 unit is defined as the amount of the enzyme that hydrolyzes 1 nmol of the RNA in 3H labeled M13 DNA/RNA hybrid to acid-soluble ribonucleotides in 20 min at 37°C. Storage: -20°C Quality: Greater than 95% protein determined by SDS-PAGE (Fig. 1, CBB staining). Endo- and exo-DNase activities and RNase activity were not detected with 100 U/ml RNaseH in 50 ul reaction at 37°C. Reagents Supplied with Enzyme: 10X RNaseH Reaction Buffer: 100 mM Tris-HCl (pH 8.0), 100 mM MgCl2, 500 mM NaCl, 10 mM DTT, 500 ug/ml BSA (Bovine Serum Albumin) Caution: To avoid contamination of trace amounts of nucleic acids in BSA, use reaction buffer that does not contain BSA and use RNaseH at higher concentrations. -
The Characterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry
Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1999 The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry. Lenore Marie Polo Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Polo, Lenore Marie, "The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry." (1999). LSU Historical Dissertations and Theses. 6923. https://digitalcommons.lsu.edu/gradschool_disstheses/6923 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter free, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. -
Supplementary Table S1 List of Proteins Identified with LC-MS/MS in the Exudates of Ustilaginoidea Virens Mol
Supplementary Table S1 List of proteins identified with LC-MS/MS in the exudates of Ustilaginoidea virens Mol. weight NO a Protein IDs b Protein names c Score d Cov f MS/MS Peptide sequence g [kDa] e Succinate dehydrogenase [ubiquinone] 1 KDB17818.1 6.282 30.486 4.1 TGPMILDALVR iron-sulfur subunit, mitochondrial 2 KDB18023.1 3-ketoacyl-CoA thiolase, peroxisomal 6.2998 43.626 2.1 ALDLAGISR 3 KDB12646.1 ATP phosphoribosyltransferase 25.709 34.047 17.6 AIDTVVQSTAVLVQSR EIALVMDELSR SSTNTDMVDLIASR VGASDILVLDIHNTR 4 KDB11684.1 Bifunctional purine biosynthetic protein ADE1 22.54 86.534 4.5 GLAHITGGGLIENVPR SLLPVLGEIK TVGESLLTPTR 5 KDB16707.1 Proteasomal ubiquitin receptor ADRM1 12.204 42.367 4.3 GSGSGGAGPDATGGDVR 6 KDB15928.1 Cytochrome b2, mitochondrial 34.9 58.379 9.4 EFDPVHPSDTLR GVQTVEDVLR MLTGADVAQHSDAK SGIEVLAETMPVLR 7 KDB12275.1 Aspartate 1-decarboxylase 11.724 112.62 3.6 GLILTLSEIPEASK TAAIAGLGSGNIIGIPVDNAAR 8 KDB15972.1 Glucosidase 2 subunit beta 7.3902 64.984 3.2 IDPLSPQQLLPASGLAPGR AAGLALGALDDRPLDGR AIPIEVLPLAAPDVLAR AVDDHLLPSYR GGGACLLQEK 9 KDB15004.1 Ribose-5-phosphate isomerase 70.089 32.491 32.6 GPAFHAR KLIAVADSR LIAVADSR MTFFPTGSQSK YVGIGSGSTVVHVVDAIASK 10 KDB18474.1 D-arabinitol dehydrogenase 1 19.425 25.025 19.2 ENPEAQFDQLKK ILEDAIHYVR NLNWVDATLLEPASCACHGLEK 11 KDB18473.1 D-arabinitol dehydrogenase 1 11.481 10.294 36.6 FPLIPGHETVGVIAAVGK VAADNSELCNECFYCR 12 KDB15780.1 Cyanovirin-N homolog 85.42 11.188 31.7 QVINLDER TASNVQLQGSQLTAELATLSGEPR GAATAAHEAYK IELELEK KEEGDSTEKPAEETK LGGELTVDER NATDVAQTDLTPTHPIR 13 KDB14501.1 14-3-3 -
Subject Index Proc
13088 Subject Index Proc. Natl. Acad. Sci. USA 91 (1994) Apoptosis in substantia nigra following developmental striatal excitotoxic Brine shrimp injury, 8117 See Artemia Visualizing hippocampal synaptic function by optical detection of Ca2l Broccol entry through the N-methyl-D-aspartate channel, 8170 See Brassica Amygdala modulation of hippocampal-dependent and caudate Bromophenacyl bromide nucleus-dependent memory processes, 8477 Bromophenacyl bromide binding to the actin-bundling protein I-plastin Distribution of corticotropin-releasing factor receptor mRNA expression inhibits inositol trisphosphate-independent increase in Ca2l in human in the rat brain and pituitary, 8777 neutrophils, 3534 Brownian dynamics Preproenkephalin promoter yields region-specific and long-term Adhesion of hard spheres under the influence of double-layer, van der expression in adult brain after direct in vivo gene transfer via a Waals, and gravitational potentials at a solid/liquid interface, 3004 defective herpes simplex viral vector, 8979 Browsers Intravenous administration of a transferrin receptor antibody-nerve Thorn-like prickles and heterophylly in Cyanea: Adaptations to extinct growth factor conjugate prevents the degeneration of cholinergic avian browsers on Hawaii?, 2810 striatal neurons in a model of Huntington disease, 9077 Bruton agammaglobulinemia Axotomy induces the expression of vasopressin receptors in cranial and Genomic organization and structure of Bruton agammaglobulinemia spinal motor nuclei in the adult rat, 9636 tyrosine kinase: Localization -
A Suppressor of a Centromere DNA Mutation Encodes a Putative Protein Kinase (MCK1)
Downloaded from genesdev.cshlp.org on October 7, 2021 - Published by Cold Spring Harbor Laboratory Press A suppressor of a centromere DNA mutation encodes a putative protein kinase (MCK1) James H. Shero 1 and Philip Hieter Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA A new approach to identify genes involved in Saccharomyces cerevisiae kinetochore function is discussed. A genetic screen was designed to recover extragenic dosage suppressors of a CEN DNA mutation. This method identified two suppressors, designated MCK1 and CMS2. Increased dosage of MCK1 specifically suppressed two similar CEN DNA mutations in CDEIII, but not comparably defective CEN DNA mutations in CDEI or CDEII. A strain containing a null allele of MCK1 was viable under standard growth conditions, had a cold-sensitive phenotype (conditional lethality at l l°C), and grew slowly on Benomyl {a microtubule-destabilizing drug). Furthermore, when grown at 18°C or in the presence of Benomyl, the null mutant exhibited a dramatic increase in the rate of mitotic chromosome loss. The allele-specific suppression and chromosome instability phenotypes suggest that MCK1 plays a role in mitotic chromosome segregation specific to CDEIII function. The MCK1 gene encodes a putative protein-serine/threonine kinase, which suggests a possible role for the MCK1 protein in regulating the activity of centromere-binding proteins by phosphorylation. MCK1 was identified and cloned independently for its involvement in the induction of meiosis and is identical to a gene that encodes a phosphotyrosyl protein with protein kinase activity. [Key Words: S. cerevisiae; kinetochore function; CEN DNA; allele-specific suppression] Received December 12, 1990; revised version accepted January 28, 1991.