DOCSLIB.ORG

Thomas Hennig

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text

Load more

Function and transport of a herpesvirus encoded Ubiquitin-specific protease in virus entry and assembly A thesis submitted to the degree of Doctor of Philosophy Imperial College London, Department of Medicine by Thomas Hennig Supervised by Professor Peter F. O’Hare Supported by Imperial College London and the Wellcome Trust September 2011 - June 2015 - 1 - Acknowledgements Acknowledgements Foremost, I would like to thank Professor Peter O’Hare for his support throughout my PhD and during the post-doctoral application process. There was not a single day when you could not ask him a question or discuss results. I think I speak for everyone in the group (and outside) in that he was always approachable, patient and that I felt well supported at every point during my PhD. The last four years working with present and past members of the group have been a great experience, allowed me to learn many new techniques and made the learning process more manageable. I even started appreciating the calming influence of classical music. I would also like to thank Dr Fernando Abaitua for his help during my first year and the very detailed discussions during the early morning hours, even after having left our group over two years ago. A big ‘thank you’ and ‘sorry’ also goes to Dr Sonia Barbosa, who could usually not escape these elaborate discussions in a timely fashion, to Dr Remi Serwa, who performed the mass spectrometry, and Nora Schmidt, who had the pleasure of reading and commenting on my thesis. I was supported by two very helpful assessors, Dr Goedele Maertens and Professor Wendy Barclay, who were always happy to advise me on any issues that arose from the work presented here. Dr Maertens kindly provided the retrovirus backbones which enabled the creation of the two inducible cell lines used for the identification of VP1-2 interacting proteins. This work would have also taken much longer without the help of Dr Anna Stockum (Dr Maertens group) who helped with the retrovirus infections. During the write-up period I also received serveral helpful comments on my thesis by my PhD examiners for which I am very grateful. There is an extensive list of additional people both at St Mary’s and outside whose help should be acknowledged here but they cannot all be listed here. I am eternally grateful for all that they have done to make my work easier and overall a very pleasant experience. I would like to thank Professor Søren Paludan for enabling a short exchange to his laboratory at Aarhus University to learn valuable skills and for discussing and supporting my application to the Wellcome Trust for further funding. Finally, I was lucky enough to be funded by the Wellcome Trust, which enabled me to present and publish some of my data and attend the laboratory of Professor Søren Paludan in Aarhus (Denmark) through provision of generous grants. Substantial additional funds towards travel and accommodation were granted by the IHW and the SGM. - 2 - Declarations Declaration of Originality I confirm that this thesis has been written and the work conducted by myself unless collaborators were acknowledged. This thesis contains the work on which I expect to be examined for the degree of Doctor of Philosophy. All work that was not conducted by myself is referenced appropriately and permissions were obtained when necessary and in accordance with Imperial College policy. Copyright declaration The copyright of this thesis rests with the author and is made available under a creative commons attribution non-commercial no derivatives licence. Researchers are free to copy, distribute or transmit that thesis on the conditions that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. - 3 - Abstract Abstract Herpes simplex virus type I (HSV-1), the prototype α-herpesvirus (HV), is a double stranded DNA virus that replicates in the nucleus of infected cells. The nuclear pore represents a gateway that must be engaged and navigated immediately after cell entry for successful infection by many classes of human viruses. For herpesviruses, capsid-tegument assemblies must be targeted to the pore where the viral genome exits and transport into the nucleus occurs. We currently have little mechanistic knowledge of this fundamental step of infection. A swath of evidence indicates that the conserved tegument protein VP1-2 is essential for early capsid transport and pore binding, and that it contains a conserved nuclear localisation signal (NLS) required for pore docking. In this thesis I undertook a detailed analysis to dissect functional determinants within the NLS from herpes simplex virus, to examine putative NLSs in VP1-2 homologues from representatives of all sub-families, to characterise protein interactions with VP1-2 and finally to construct a GFP expressing entry defective recombinant virus to study the consequences of infection. I show that the HSV NLS can function as a mono-or bipartite motif and has a particular organisation conserved in the -herpesvirus homologues but distinct from those in the - and - herpesviruses. The representatives of all 3 classes contain a functional NLS at approximately the same position. All bi-partite motifs were able to rescue the HSV VP1-2ΔNLS virus defect albeit to varying extent whereas the mono-partite HHV-8 motif did not. I constructed and purified chimeric recombinant viruses for the VZV, HCMV and EBV motifs and show distinct differences in their ability to replicate in non-complementing cells. In HSV, NLS function in the context of protein nuclear import or of viable virus replication, was dependent on lysine 428 and the integrity of the full bi-partite motif. Mutations which reduced NLS activity generally caused reduced fitness of recombinant viruses. For the analysis of interaction partners of VP1-2 and the NLS, I developed a one-step approach to analyse the capsid interactome during entry. Additionally, I constructed mammalian GST-VP1-2.NLS fusion proteins and cell lines which inducibly express the N-terminal region of VP1-2 for analysis of NLS interacting proteins. Using mass spectrometry (MS) I identified a number of VP1-2-interacting, cellular proteins including DTX3L, an important regulator of the DNA damage response. - 4 - Abbreviations Abbreviations BES N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid BHV-1 bovine Herpesvirus-1 BoHV-4 bovine Herpesvirus-4 bpNLS bi-partite NLS BSA bovine serum albumin CA Capsid protein (of HIV) cNLS classical NLS CPE cytopathic effect CV Crystal violet DAB Diaminobenzidine DABCO diazabicyclooctane DAPI 4′,6-Diamidin-2-phenylindol ddH2O double distilled H2O DDR DNA damage response DMEM Dulbecco's Modified Eagle's Medium DMSO dimethyl sulfoxide dsDNA/RNA double stranded DNA/RNA EBV Epstein Barr Virus EHV equine Herpesvirus EM electron microscopy ER endoplasmatic reticulum ERAD ER associated protein degradation FBS fetal bovine serum GAG glycosaminoglycan gD (or similar) HV glycoprotein D GST Glutathione-S transferase HA hemagglutinin HBS HEPES buffered saline HCMV human Cytomegalovirus HHV human Herpesvirus HIV human immunodeficiency virus HP1 heterochromatin protein 1 HR homologous recombination HRP horseradish peroxidase HS human serum HSV herpes simplex virus HV herpesvirus HVEM HV entry mediator HVS Herpesvirus Saimiri IAV influenza A virus IBB Importin-β binding domain ICP infected cell protein IN integrase (of HIV) INM inner nuclear membrane - 5 - Abbreviations IP immunoprecipitation ISG interferon stimulated genes lTag large T antigen (of SV40) M1/2 matrix protein 1/2 (of IAV) MCS multiple cloning site MLV murine leukemia virus MOI multiplicity of infection mpNLS mono-partite NSL MS mass spectrometry MTOC microtubule-organising centre MuHV murid Herpesvirus MyoNLS NLS of Myopodin NCS new-born calf serum NE nuclear envelope NEAA non-essential amino acids NEBD nuclear envelope breakdown NES nuclear export signal NHEJ non-homologous end joining NLS nuclear localisation signal NPC nuclear pore complex NP nucleoprotein (of IAV) Nups Nucleoproteins ONM outer nuclear membrane ORF open reading frame PAA phosphono acetic acid PBS phosphate buffered saline Pen/Strep Penicillin/Streptomycin PFA paraformaldehyde PFU plaque forming unit PIC pre-integration complex (of HIV) PMSF Phenylmethanesulfonyl fluoride PRV Pseudorabies Virus pUL37 protein product of open reading frame 37 of unique long genome segment pUS3 protein product of open reading frame 3 of the unique short genome segment SDS sodium dodecyl sulfate SDS-PAGE SDS polyacrylamide gel electropophoresis ssDNA/RNA single stranded DNA/RNA SV40 Simian virus 40 TGN trans Golgi network ts mutant temperature-sensitive mutant USP ubiquitin-specific protease Vhs virion host shutoff protein VP virion protein Vpr viral protein R (of HIV) VZV Varicella zoster virus β-gal β-galactosidase - 6 - Table of contents 1 INTRODUCTION ................................................................................................... - 16 - 1.1 Introduction to herpesviruses ............................................................................................... - 16 - 1.1.1 Discovery and classification of Herpesviruses .................................................................... - 16 - 1.1.2 Introduction to Herpes Simplex Virus ................................................................................ - 17 - 1.1.3 Virion structure..................................................................................................................
Recommended publications
  • Transportin 1 Accumulates Specifically with FET Proteins but No Other

    Transportin 1 Accumulates Specifically with FET Proteins but No Other

    CORE Metadata, citation and similar papers at core.ac.uk Provided by RERO DOC Digital Library Acta Neuropathol DOI 10.1007/s00401-012-1020-6 ORIGINAL PAPER Transportin 1 accumulates specifically with FET proteins but no other transportin cargos in FTLD-FUS and is absent in FUS inclusions in ALS with FUS mutations Manuela Neumann • Chiara F. Valori • Olaf Ansorge • Hans A. Kretzschmar • David G. Munoz • Hirofumi Kusaka • Osamu Yokota • Kenji Ishihara • Lee-Cyn Ang • Juan M. Bilbao • Ian R. A. Mackenzie Received: 22 May 2012 / Accepted: 16 July 2012 Ó Springer-Verlag 2012 Abstract Accumulation of the DNA/RNA binding pro- in these conditions. While ALS-FUS showed only accu- tein fused in sarcoma (FUS) as inclusions in neurons and mulation of FUS, inclusions in FTLD-FUS revealed co- glia is the pathological hallmark of amyotrophic lateral accumulation of all members of the FET protein family, that sclerosis patients with mutations in FUS (ALS-FUS) as well include FUS, Ewing’s sarcoma (EWS) and TATA-binding as in several subtypes of frontotemporal lobar degeneration protein-associated factor 15 (TAF15) suggesting a more (FTLD-FUS), which are not associated with FUS muta- complex disturbance of transportin-mediated nuclear tions. Despite some overlap in the phenotype and import of proteins in FTLD-FUS compared to ALS-FUS. neuropathology of FTLD-FUS and ALS-FUS, significant To gain more insight into the mechanisms of inclusion body differences of potential pathomechanistic relevance were formation, we investigated the role of Transportin 1 (Trn1) recently identified in the protein composition of inclusions as well as 13 additional cargo proteins of Transportin in the spectrum of FUS-opathies by immunohistochemistry and biochemically.
  • Dynamics of Gene Silencing During X Inactivation Using Allele-Specific RNA-Seq Hendrik Marks1*, Hindrik H

    Dynamics of Gene Silencing During X Inactivation Using Allele-Specific RNA-Seq Hendrik Marks1*, Hindrik H

    Marks et al. Genome Biology (2015) 16:149 DOI 10.1186/s13059-015-0698-x RESEARCH Open Access Dynamics of gene silencing during X inactivation using allele-specific RNA-seq Hendrik Marks1*, Hindrik H. D. Kerstens1, Tahsin Stefan Barakat3, Erik Splinter4, René A. M. Dirks1, Guido van Mierlo1, Onkar Joshi1, Shuang-Yin Wang1, Tomas Babak5, Cornelis A. Albers2, Tüzer Kalkan6, Austin Smith6, Alice Jouneau7, Wouter de Laat4, Joost Gribnau3 and Hendrik G. Stunnenberg1* Abstract Background: During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results: Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs.
  • Identification of the Binding Partners for Hspb2 and Cryab Reveals

    Identification of the Binding Partners for Hspb2 and Cryab Reveals

    Brigham Young University BYU ScholarsArchive Theses and Dissertations 2013-12-12 Identification of the Binding arP tners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non- Redundant Roles for Small Heat Shock Proteins Kelsey Murphey Langston Brigham Young University - Provo Follow this and additional works at: https://scholarsarchive.byu.edu/etd Part of the Microbiology Commons BYU ScholarsArchive Citation Langston, Kelsey Murphey, "Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins" (2013). Theses and Dissertations. 3822. https://scholarsarchive.byu.edu/etd/3822 This Thesis is brought to you for free and open access by BYU ScholarsArchive. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of BYU ScholarsArchive. For more information, please contact [email protected], [email protected]. Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactions and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston A thesis submitted to the faculty of Brigham Young University in partial fulfillment of the requirements for the degree of Master of Science Julianne H. Grose, Chair William R. McCleary Brian Poole Department of Microbiology and Molecular Biology Brigham Young University December 2013 Copyright © 2013 Kelsey Langston All Rights Reserved ABSTRACT Identification of the Binding Partners for HspB2 and CryAB Reveals Myofibril and Mitochondrial Protein Interactors and Non-Redundant Roles for Small Heat Shock Proteins Kelsey Langston Department of Microbiology and Molecular Biology, BYU Master of Science Small Heat Shock Proteins (sHSP) are molecular chaperones that play protective roles in cell survival and have been shown to possess chaperone activity.
  • Efficacy and Mechanistic Evaluation of Tic10, a Novel Antitumor Agent

    Efficacy and Mechanistic Evaluation of Tic10, a Novel Antitumor Agent

    University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2012 Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent Joshua Edward Allen University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Oncology Commons Recommended Citation Allen, Joshua Edward, "Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent" (2012). Publicly Accessible Penn Dissertations. 488. https://repository.upenn.edu/edissertations/488 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/488 For more information, please contact [email protected]. Efficacy and Mechanisticv E aluation of Tic10, A Novel Antitumor Agent Abstract TNF-related apoptosis-inducing ligand (TRAIL; Apo2L) is an endogenous protein that selectively induces apoptosis in cancer cells and is a critical effector in the immune surveillance of cancer. Recombinant TRAIL and TRAIL-agonist antibodies are in clinical trials for the treatment of solid malignancies due to the cancer-specific cytotoxicity of TRAIL. Recombinant TRAIL has a short serum half-life and both recombinant TRAIL and TRAIL receptor agonist antibodies have a limited capacity to perfuse to tissue compartments such as the brain, limiting their efficacy in certain malignancies. To overcome such limitations, we searched for small molecules capable of inducing the TRAIL gene using a high throughput luciferase reporter gene assay. We selected TRAIL-inducing compound 10 (TIC10) for further study based on its induction of TRAIL at the cell surface and its promising therapeutic index. TIC10 is a potent, stable, and orally active antitumor agent that crosses the blood-brain barrier and transcriptionally induces TRAIL and TRAIL-mediated cell death in a p53-independent manner.
  • Mouse Get4 Knockout Project (CRISPR/Cas9)

    Mouse Get4 Knockout Project (CRISPR/Cas9)

    https://www.alphaknockout.com Mouse Get4 Knockout Project (CRISPR/Cas9) Objective: To create a Get4 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Get4 gene (NCBI Reference Sequence: NM_026269 ; Ensembl: ENSMUSG00000025858 ) is located on Mouse chromosome 5. 9 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 9 (Transcript: ENSMUST00000026976). Exon 2~8 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Exon 2 starts from about 15.9% of the coding region. Exon 2~8 covers 75.43% of the coding region. The size of effective KO region: ~4995 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 5 6 7 8 9 Legends Exon of mouse Get4 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1505 bp section downstream of Exon 8 is aligned with itself to determine if there are tandem repeats.
  • Focus on the Small Heat Shock Protein HSPB1 Autofagie in De Erfelij

    Focus on the Small Heat Shock Protein HSPB1 Autofagie in De Erfelij

    Faculteit Faculteit Farmaceutische, Biomedische en Diergeneeskundige wetenschappen Biochemie en Biotechnologie Autophagy in inherited peripheral neuropathies: Focus on the small heat shock protein HSPB1 Autofagie in de erfelijke perifere neuropathieën: Focus op de kleine heat shock proteïne HSPB1 Proefschrift voorgelegd tot het behalen van de graad van Doctor in de Wetenschappen: Biochemie en Biotechnologie aan de Universiteit Antwerpen. te verdedigen door Mansour HAIDAR Promotor Prof. Dr. Vincent Timmerman Antwerpen, 2018 1 2 “Haud igitur redit ad Nihilum res ulla, sed omnes Discidio redeunt in corpora materiai” Lucretius, De Rerum Natura, Book I. 250 3 4 Members of the jury Chair Prof. Dr. Wim Vanden Berghe, PhD (UA, Antwerp, Belgium) Promotor Prof. Dr. Vincent Timmerman, PhD (UA, Antwerp, Belgium) Internal jury member Prof. Dr. Wim Martinet, PhD (UA, Antwerp, Belgium) External jury members Prof. Dr. Joy Irobi (UHasselt, Hasselt, Belgium) Prof. Dr. Maurizio D’Antonio (San Raffaele Institute, Milan, Italy) Prof. Dr. Ir. Winnok De Vos (UA, Antwerp, Belgium) 5 6 Table of Contents Summary/Samenvatting 9 Rationale and Aims 13 Introduction Chapter 1 Autophagy as an emerging common pathomechanism in inherited 15 peripheral neuropathies Chapter 2 Small heat shock proteins: Their role in proteostasis 79 and neurodegeneration Results Chapter 3 HSPB1 is required for Autophagy: Insights from CMT-causing mutations 103 Chapter 4 An interactomics study of HSPB1 wild-type and mutant links it to the 129 autophagy receptor P62 Discussion 179 List of abbreviations 195 Curriculum Vitae 199 Acknowledgements 203 7 8 Summary Inherited peripheral neuropathies (IPNs) are genetically heterogeneous disorders affecting mainly the peripheral nervous system and with over 1500 mutations in more than 80 affected genes discovered so far.
  • FUS-NLS/Transportin 1 Complex Structure Provides

    FUS-NLS/Transportin 1 Complex Structure Provides

    University of Kentucky UKnowledge Molecular and Cellular Biochemistry Faculty Molecular and Cellular Biochemistry Publications 10-8-2012 FUS-NLS/Transportin 1 complex structure provides insights into the nuclear targeting mechanism of FUS and the implications in ALS Chunyan Niu Chinese Academy of Sciences, China Jiayu Zhang University of Kentucky, [email protected] Feng Gao Chinese Academy of Sciences, China Liuqing Yang University of Kentucky, [email protected] Minze Jia Chinese Academy of Sciences, China See next page for additional authors Right click to open a feedback form in a new tab to let us know how this document benefits oy u. Follow this and additional works at: https://uknowledge.uky.edu/biochem_facpub Part of the Biochemistry, Biophysics, and Structural Biology Commons Repository Citation Niu, Chunyan; Zhang, Jiayu; Gao, Feng; Yang, Liuqing; Jia, Minze; Zhu, Haining; and Gong, Weimin, "FUS-NLS/Transportin 1 complex structure provides insights into the nuclear targeting mechanism of FUS and the implications in ALS" (2012). Molecular and Cellular Biochemistry Faculty Publications. 9. https://uknowledge.uky.edu/biochem_facpub/9 This Article is brought to you for free and open access by the Molecular and Cellular Biochemistry at UKnowledge. It has been accepted for inclusion in Molecular and Cellular Biochemistry Faculty Publications by an authorized administrator of UKnowledge. For more information, please contact [email protected]. Authors Chunyan Niu, Jiayu Zhang, Feng Gao, Liuqing Yang, Minze Jia, Haining Zhu, and Weimin Gong FUS-NLS/Transportin 1 complex structure provides insights into the nuclear targeting mechanism of FUS and the implications in ALS Notes/Citation Information Published in PLoS ONE, v.
  • Supporting Information

    Supporting Information

    Supporting Information Edgar et al. 10.1073/pnas.1601895113 SI Methods (Actimetrics), and recordings were analyzed using LumiCycle Mice. Sample size was determined using the resource equation: Data Analysis software (Actimetrics). E (degrees of freedom in ANOVA) = (total number of exper- – Cell Cycle Analysis of Confluent Cell Monolayers. NIH 3T3, primary imental animals) (number of experimental groups), with −/− sample size adhering to the condition 10 < E < 20. For com- WT, and Bmal1 fibroblasts were sequentially transduced − − parison of MuHV-4 and HSV-1 infection in WT vs. Bmal1 / with lentiviral fluorescent ubiquitin-based cell cycle indicators mice at ZT7 (Fig. 2), the investigator did not know the genotype (FUCCI) mCherry::Cdt1 and amCyan::Geminin reporters (32). of the animals when conducting infections, bioluminescence Dual reporter-positive cells were selected by FACS (Influx Cell imaging, and quantification. For bioluminescence imaging, Sorter; BD Biosciences) and seeded onto 35-mm dishes for mice were injected intraperitoneally with endotoxin-free lucif- subsequent analysis. To confirm that expression of mCherry:: Cdt1 and amCyan::Geminin correspond to G1 (2n DNA con- erin (Promega E6552) using 2 mg total per mouse. Following < ≤ anesthesia with isofluorane, they were scanned with an IVIS tent) and S/G2 (2 n 4 DNA content) cell cycle phases, Lumina (Caliper Life Sciences), 15 min after luciferin admin- respectively, cells were stained with DNA dye DRAQ5 (abcam) and analyzed by flow cytometry (LSR-Fortessa; BD Biosci- istration. Signal intensity was quantified using Living Image ences). To examine dynamics of replicative activity under ex- software (Caliper Life Sciences), obtaining maximum radiance perimental confluent conditions, synchronized FUCCI reporter for designated regions of interest (photons per second per − − − monolayers were observed by time-lapse live cell imaging over square centimeter per Steradian: photons·s 1·cm 2·sr 1), relative 3 d (Nikon Eclipse Ti-E inverted epifluorescent microscope).
  • Genetic Variant in 3' Untranslated Region of the Mouse Pycard Gene

    Genetic Variant in 3' Untranslated Region of the Mouse Pycard Gene

    bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437184; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 Title: 4 Genetic Variant in 3’ Untranslated Region of the Mouse Pycard Gene Regulates Inflammasome 5 Activity 6 Running Title: 7 3’UTR SNP in Pycard regulates inflammasome activity 8 Authors: 9 Brian Ritchey1*, Qimin Hai1*, Juying Han1, John Barnard2, Jonathan D. Smith1,3 10 1Department of Cardiovascular & Metabolic Sciences, Lerner Research Institute, Cleveland Clinic, 11 Cleveland, OH 44195 12 2Department of Quantitative Health Sciences, Lerner Research Institute, Cleveland Clinic, Cleveland, OH 13 44195 14 3Department of Molecular Medicine, Cleveland Clinic Lerner College of Medicine of Case Western 15 Reserve University, Cleveland, OH 44195 16 *, These authors contributed equally to this study. 17 Address correspondence to Jonathan D. Smith: email [email protected]; ORCID ID 0000-0002-0415-386X; 18 mailing address: Cleveland Clinic, Box NC-10, 9500 Euclid Avenue, Cleveland, OH 44195, USA. 19 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.26.437184; this version posted March 26, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 20 Abstract 21 Quantitative trait locus mapping for interleukin-1 release after inflammasome priming and activation 22 was performed on bone marrow-derived macrophages (BMDM) from an AKRxDBA/2 strain intercross.
  • Nucleolin and Its Role in Ribosomal Biogenesis

    Nucleolin and Its Role in Ribosomal Biogenesis

    NUCLEOLIN: A NUCLEOLAR RNA-BINDING PROTEIN INVOLVED IN RIBOSOME BIOGENESIS Inaugural-Dissertation zur Erlangung des Doktorgrades der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf vorgelegt von Julia Fremerey aus Hamburg Düsseldorf, April 2016 2 Gedruckt mit der Genehmigung der Mathematisch-Naturwissenschaftlichen Fakultät der Heinrich-Heine-Universität Düsseldorf Referent: Prof. Dr. A. Borkhardt Korreferent: Prof. Dr. H. Schwender Tag der mündlichen Prüfung: 20.07.2016 3 Die vorgelegte Arbeit wurde von Juli 2012 bis März 2016 in der Klinik für Kinder- Onkologie, -Hämatologie und Klinische Immunologie des Universitätsklinikums Düsseldorf unter Anleitung von Prof. Dr. A. Borkhardt und in Kooperation mit dem ‚Laboratory of RNA Molecular Biology‘ an der Rockefeller Universität unter Anleitung von Prof. Dr. T. Tuschl angefertigt. 4 Dedicated to my family TABLE OF CONTENTS 5 TABLE OF CONTENTS TABLE OF CONTENTS ............................................................................................... 5 LIST OF FIGURES ......................................................................................................10 LIST OF TABLES .......................................................................................................12 ABBREVIATION .........................................................................................................13 ABSTRACT ................................................................................................................19 ZUSAMMENFASSUNG
  • SLIC-CAGE: High-Resolution Transcription Start Site Mapping Using Nanogram-Levels of Total RNA

    SLIC-CAGE: High-Resolution Transcription Start Site Mapping Using Nanogram-Levels of Total RNA

    bioRxiv preprint doi: https://doi.org/10.1101/368795; this version posted July 19, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license. SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA Nevena Cvetesic1, 2*, Harry G. Leitch1,2, Malgorzata Borkowska1,2, Ferenc Müller3, Piero Carninci4,5, Petra Hajkova1,2 and Boris Lenhard1,2,6* 1Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London W12 0NN, UK 2MRC London Institute of Medical Sciences, London W12 0NN, UK 3Institute of Cancer and Genomic Sciences, University of Birmingham, Edgbaston B15 2TT, UK 4RIKEN Center for Life Science Technologies, Division of Genomic Technologies, Yokohama City, Kanagawa 230-0045, Japan 5RIKEN Omics Science Center, Yokohama City, Kanagawa 230-0045, Japan 6Sars International Centre for Marine Molecular Biology, University of Bergen, N-5008 Bergen, Norway *Correspondence to: Nevena Cvetesic Institute of Clinical Sciences, Faculty of Medicine, Imperial College London and MRC London Institute of Medical Sciences, London, W12 0NN, UK e-mail: [email protected] Boris Lenhard Institute of Clinical Sciences, Faculty of Medicine, Imperial College London and MRC London Institute of Medical Sciences, London, W12 0NN, UK e-mail: [email protected] Running title: TSS discovery using Super-Low Input Carrier-CAGE Keywords: cap analysis of gene expression; low input material; degradable nucleic acid carrier; promoters; gene expression profiling 1 bioRxiv preprint doi: https://doi.org/10.1101/368795; this version posted July 19, 2018.
  • Emergence of the Noncoding Cancer Genome: a Target of Genetic and Epigenetic Alterations

    Emergence of the Noncoding Cancer Genome: a Target of Genetic and Epigenetic Alterations

    Published OnlineFirst October 19, 2016; DOI: 10.1158/2159-8290.CD-16-0745 REVIEW Emergence of the Noncoding Cancer Genome: A Target of Genetic and Epigenetic Alterations Stanley Zhou 1 , 2 , Aislinn E. Treloar 1 , 2 , and Mathieu Lupien 1 , 2 , 3 ABSTRACT The emergence of whole-genome annotation approaches is paving the way for the comprehensive annotation of the human genome across diverse cell and tissue types exposed to various environmental conditions. This has already unmasked the positions of thou- sands of functional cis-regulatory elements integral to transcriptional regulation, such as enhancers, promoters, and anchors of chromatin interactions that populate the noncoding genome. Recent studies have shown that cis-regulatory elements are commonly the targets of genetic and epigenetic altera- tions associated with aberrant gene expression in cancer. Here, we review these fi ndings to showcase the contribution of the noncoding genome and its alteration in the development and progression of can- cer. We also highlight the opportunities to translate the biological characterization of genetic and epi- genetic alterations in the noncoding cancer genome into novel approaches to treat or monitor disease. Signifi cance: The majority of genetic and epigenetic alterations accumulate in the noncoding genome throughout oncogenesis. Discriminating driver from passenger events is a challenge that holds great promise to improve our understanding of the etiology of different cancer types. Advancing our under- standing of the noncoding cancer genome may thus identify new therapeutic opportunities and acceler- ate our capacity to fi nd improved biomarkers to monitor various stages of cancer development. Cancer Discov; 6(11); 1215–29.