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Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations Fall 2010 Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors Bart R. Anderson University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Molecular Biology Commons Recommended Citation Anderson, Bart R., "Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors" (2010). Publicly Accessible Penn Dissertations. 1567. https://repository.upenn.edu/edissertations/1567 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1567 For more information, please contact [email protected]. Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors Abstract Multiple innate defense pathways exist to recognize and defend against foreign nucleic acids. Unlike innate immune receptors that recognize structures specific for pathogens that are not shared by mammalian hosts — for example, toll-like receptor (TLR)4-lipopolysaccharide, TLR5-flagellin, NOD1 and 2-peptidoglycan — all nucleic acids are made from four components that are identical from bacteria to man. Nucleoside modifications are prevalent in nature but vary greatly in their distribution and frequency, and therefore could serve as patterns for recognition of pathogenic nucleic acids. The presence of modified nucleosides in RNA reduces the activation of RNA-sensing TLRs and retinoic acid inducible gene I (RIG-I), which initiate signaling cascades following activation and result in transcription of pro- inflammatory genes. Unexpectedly, translation of in vitro transcribed mRNA is enhanced by incorporation of modified nucleosides, but the mechanism esponsibler for this enhanced translation has not been identified. oT identify the pathways responsible for enhanced translation of modified nucleoside- containing mRNA, we studied two cytoplasmic RNA-sensing innate defense mechanisms known to influence translation, the RNA-dependent protein kinase (PKR) pathway and the 2-5A system (oligoadenylate synthetase [OAS] and RNase L). Using purified protein in vitro, cell culture, and in vivo mouse studies, we show that unmodified in vitro transcribed mRNA activates PKR and OAS and is rapidly cleaved by RNase L. However, we show that incorporation of modified nucleosides into in vitro transcribed mRNA reduces each of these pathways. Furthermore, we demonstrate that these pathways are necessary for enhanced translation of mRNA containing modified nucleosides. Additionally, we demonstrate that the presence of pseudouridine in in vitro transcripts increases mRNA half-life following delivery. From these data, we conclude that unmodified in vitro transcribed mRNA is stimulatory to the cytoplasmic RNA sensors PKR and OAS. This stimulation is reduced by the presence of modified nucleosides. The enhanced translation of mRNA containing modified nucleosides esultsr from reduced PKR and OAS activation. These data support a larger interpretation that the absence or reduction in frequency of modified nucleosides in RNA is a common pattern for recognition of pathogenic RNA by numerous innate defense systems. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Drew Weissman Keywords RNA-dependent protein kinase R (PKR), Oligoadenylate synthetase (OAS), Ribonuclease L (RNase L), pseudouridine, innate immunity, nucleofector Subject Categories Molecular Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1567 NUCLEOSIDE MODIFICATIONS SUPPRESS RNA ACTIVATION OF CYTOPLASMIC RNA SENSORS Bart R. Anderson A DISSERTATION in Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2010 Supervisor of Dissertation _______________________________ Drew Weissman, M.D., Ph.D. Associate Professor of Medicine Graduate Group Chairperson _______________________________ Daniel S. Kessler, Ph.D. Associate Professor of Cell and Developmental Biology Dissertation Committee Jean Bennett, M.D., Ph.D., F.M. Kirby Professor of Ophthalmology James H. Eberwine, Ph.D., Elmer Holmes Bobst Professor of Pharmacology Alan M. Gewirtz, M.D., C. Willard Robinson Professor of Hematology-Oncology Zissimos Mourelatos, M.D., Associate Professor of Pathology and Laboratory Medicine Nucleoside Modifications Suppress RNA Activation of Cytoplasmic RNA Sensors © COPYRIGHT 2010 Bart Russell Anderson Dedication First and foremost this work is a tribute to my wonderful wife, Katie Anderson. She cheerfully and tirelessly provided the patient encouragement and loving support which were critical to completing this effort. She believes in me even when I do not believe in myself, and helps me be who she believes I am. The numberless hours she spent listening to presentations and editing my writing count double, knowing that science itself holds little interest and her only goal has been my success. I cannot express how much her sustaining assistance means to me. I am also grateful for the love and interest of my family: my mother, Julianne Anderson, my siblings, Richelle, Shawn, Derek, Kurt, and Ashley, as well as my in-laws. They are always interested in my development and excited with my successes, and are integral to who I am. Finally, I dedicate a special thanks to my father, Russell Anderson. Although he did not live to see the completion of this work, I know he would have been proud. His brilliant mind and love of learning instilled within me a joy in understanding and discovery that is the foundation of my passion for research. First as a physician and then as a patient participant in clinical trials, he believed in the value of medicine and science to preserve and improve lives. If research can produce insights that preserve a life like his then we can call science a success. iii Acknowledgements First I owe a debt of gratitude to Drew Weissman. He has been an excellent mentor and advisor. I similarly have been guided greatly by Kati Karikó, who guided me closely throughout this work and contributed significantly to my development. I thank lab members who encouraged and supported me, including Jeremy Wingard, Hiromi Muramatsu, Houping Ni, Haitao Hu, Yu Zhou, Klara Balint, Veronica Holmes, Earl Stoddard and Kathy Fernando. I would like to thank my thesis committee, Alan Gewirtz, Jim Eberwine, Jean Bennett, and Zissimos Mourelatos for their guidance, insights, and suggestions throughout my dissertation research. Within the gene therapy and vaccines group administration, I also thank Jane Glick, Jim Wilson, and Dave Weiner for advice and guidance. I am grateful to Philip Bevilacqua and his lab at Pennsylvania State University, in particular Rao Nallagatla, for training me in techniques and for generously supplying reagents for PKR experiments. I appreciate the generosity of Robert Silverman and his lab, especially Babel Jha, for hosting me at the Cleveland Clinic, training me in OAS and RNase L experiments, and for providing reagents and cell lines. Tom Dever (NIH) provided plasmids encoding PKR inhibitor proteins. David Ron (NYU) and Alan Diehl (UPenn) (with permission from Douglas Cavener, PSU) provided MEF cell lines for nucleofection experiments. VIRxSYS Corporation (Xiaobin Lu) provided the lentiviral vector. The following reagents were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 consensus subtype C env (15- mer) peptides – Complete Set; HIV-1 consensus subtype B gag peptides – Complete Set; iv and HIV-1 p24 monoclonal antibody (183-H12-5C) from Dr. Bruce Chesebro and Kathy Wehrly. I was supported by NIH Cell & molecular biology training grant T32DK07748, NIH Gene therapy training grant T32GM07229, and NIH Comparative medical & molecular genetics training grant T32RR007063. This work was also supported by National Institutes of Health grants R01AI50484, R21DE019059, and AI-50484 to Drew Weissman and R42HL87688 to Katalin Karikó. v ABSTRACT NUCLEOSIDE MODIFICATIONS SUPPRESS RNA ACTIVATION OF CYTOPLASMIC RNA SENSORS Bart R. Anderson Dissertation Supervisor: Drew Weissman, M.D., Ph.D. Multiple innate defense pathways exist to recognize and defend against foreign nucleic acids. Unlike innate immune receptors that recognize structures specific for pathogens that are not shared by mammalian hosts — for example, toll-like receptor (TLR)4- lipopolysaccharide, TLR5-flagellin, NOD1 and 2-peptidoglycan — all nucleic acids are made from four components that are identical from bacteria to man. Nucleoside modifications are prevalent in nature but vary greatly in their distribution and frequency, and therefore could serve as patterns for recognition of pathogenic nucleic acids. The presence of modified nucleosides in RNA reduces the activation of RNA-sensing TLRs and retinoic acid inducible gene I (RIG-I), which initiate signaling cascades following activation and result in transcription of pro-inflammatory genes. Unexpectedly, translation of in vitro transcribed mRNA is enhanced by incorporation of modified nucleosides, but the mechanism responsible for this enhanced translation has not been identified. To identify the pathways responsible for enhanced translation of modified nucleoside-containing mRNA, we studied two cytoplasmic RNA-sensing innate defense vi mechanisms known to influence translation, the RNA-dependent
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