title: The role of sequence context, nucleotide pool balance and stress in 2'-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA.
abstract: The misincorporation of 2'-deoxyribonucleotides (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fidelity of coding RNAs and the mutagenic evolution of viral RNA genomes. However, quantitative appreciation for the degree to which dN misincorporation occurs is limited by the lack of analytical tools. Here, we report a method to hydrolyze RNA to release 2'-deoxyribonucleotide-ribonucleotide pairs (dNrN) that are then quantified by chromatography-coupled mass spectrometry (LC-MS). Using this platform, we found misincorporated dNs occurring at 1 per 103to 105ribonucleotide (nt) in mRNA, rRNAs and tRNA in human cells, Escherichia coli, Saccharomyces cerevisiae and, most abundantly, in the RNA genome of dengue virus. The frequency of dNs varied widely among organisms and sequence contexts, and partly reflected the in vitro discrimination efficiencies of different RNA polymerases against 2'-deoxyribonucleoside 5'-triphosphates (dNTPs). Further, we demonstrate a strong link between dN frequencies in RNA and the balance of dNTPs and ribonucleoside 5'-triphosphates (rNTPs) in the cellular pool, with significant stress-induced variation of dN incorporation. Potential implications of dNs in RNA are discussed, including the possibilities of dN incorporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune defense mechanism against viral infections.
authors.affiliation: Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602.
authors.forename: Jin
authors.initials: J
authors.lastname: Wang
authors.affiliation: Novartis Institute for Tropical Diseases, Singapore 138670.
authors.forename: Hongping
authors.initials: H
authors.lastname: Dong
authors.affiliation: Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602 Department of Microbiology & Immunology Programme, Center for Life Sciences, National University of Singapore, Singapore 117545.
authors.forename: Yok Hian
authors.initials: YH
authors.lastname: Chionh
authors.affiliation: Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602.
authors.forename: Megan E
authors.initials: ME
authors.lastname: McBee
authors.affiliation: Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602.
authors.forename: Sasilada
authors.initials: S
authors.lastname: Sirirungruang
authors.affiliation: Department of Biological Sciences, The University at Albany, Albany, NY 12222, USA.
authors.forename: Richard P
authors.initials: RP
authors.lastname: Cunningham
authors.affiliation: Departments of Biochemistry & Molecular Biology and Phamarcology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
authors.forename: Pei-Yong
authors.initials: PY
authors.lastname: Shi
authors.affiliation: Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602 Department of Biological Engineering & Center for Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA pcdedon@mit.edu.
authors.forename: Peter C
authors.initials: PC
authors.lastname: Dedon
mesh_terms: D000818:Animals; D001482:Base Composition; D002460:Cell Line; D002853:Chromatography, Liquid; D003854:Deoxyribonucleotides / Q000737:chemistry*; D005057:Eukaryotic Cells / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008322:Mammals; D016296:Mutagenesis; D011387:Prokaryotic Cells / Q000378:metabolism; D012313:RNA / Q000737:chemistry* / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry / Q000235:genetics; D012265:Ribonucleotides; D013312:Stress, Physiological / Q000235:genetics*; D053719:Tandem Mass Spectrometry
chemical_list: D003854:Deoxyribonucleotides; D012329:RNA, Bacterial; D012367:RNA, Viral; D012265:Ribonucleotides; D012313:RNA