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The Role of Sequence Context, Nucleotide Pool Balance and Stress in 2#-Deoxynucleotide Misincorporation in Viral, Bacterial and Mammalian RNA

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The Role of Sequence Context, Nucleotide Pool Balance and Stress in 2#-Deoxynucleotide Misincorporation in Viral, Bacterial and Mammalian RNA The role of sequence context, nucleotide pool balance and stress in 2#-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Wang, Jin et al. “The Role of Sequence Context, Nucleotide Pool Balance and Stress in 2#-Deoxynucleotide Misincorporation in Viral, Bacterial and Mammalian RNA.” Nucleic Acids Research 44.18 (2016): 8962–8975. As Published http://dx.doi.org/10.1093/nar/gkw572 Publisher Oxford University Press Version Final published version Citable link http://hdl.handle.net/1721.1/106823 Terms of Use Creative Commons Attribution 4.0 International License Detailed Terms http://creativecommons.org/licenses/by/4.0/ Nucleic Acids Research Advance Access published June 30, 2016 Nucleic Acids Research, 2016 1 doi: 10.1093/nar/gkw572 The role of sequence context, nucleotide pool balance and stress in 2-deoxynucleotide misincorporation in viral, bacterial and mammalian RNA Jin Wang1, Hongping Dong2, Yok Hian Chionh1,3, Megan E. McBee1, Sasilada Sirirungruang1, Richard P. Cunningham4, Pei-Yong Shi5 and Peter C. Dedon1,6,* 1Infectious Disease Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore 138602, 2Novartis Institute for Tropical Diseases, Singapore 138670, 3Department of Microbiology & Immunology Programme, Center for Life Sciences, National University of Singapore, Singapore 117545, 4Department of Biological Sciences, The University at Albany, Albany, NY 12222, USA, 5Departments of Biochemistry & Molecular Biology and Phamarcology & Toxicology, and Sealy Center for Structural Biology & Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA and 6Department of Biological Engineering & Center for Downloaded from Environmental Health Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139-4307, USA Received April 10, 2015; Revised June 05, 2016; Accepted June 06, 2016 http://nar.oxfordjournals.org/ ABSTRACT corporation in RNA as a contributing factor in viral evolution and human disease, and as a host immune The misincorporation of 2 -deoxyribonucleotides defense mechanism against viral infections. (dNs) into RNA has important implications for the function of non-coding RNAs, the translational fi- INTRODUCTION delity of coding RNAs and the mutagenic evolu- tion of viral RNA genomes. However, quantitative The potential for misincorporation of ribonucleotides (rN) appreciation for the degree to which dN misincor- into DNA and 2 -deoxyribonucleotides (dN) into RNA has poration occurs is limited by the lack of analytical long been recognized as a possible consequence of the abil- at MIT Libraries on October 6, 2016 tools. Here, we report a method to hydrolyze RNA to ity of polymerases to discriminate among components of the highly dynamic nucleotide pool in prokaryotic and eu- release 2 -deoxyribonucleotide-ribonucleotide pairs karyotic cells in different environments (1–3). A key dis- (dNrN) that are then quantified by chromatography- criminatory feature here is the 2-OHinRNAthatplays coupled mass spectrometry (LC-MS). Using this plat- critical roles in protein–RNA recognition (4), stabilization form, we found misincorporated dNs occurring at 1 of RNA structures (5–7), RNA packing (8) and RNA- per 103 to 105 ribonucleotide (nt) in mRNA, rRNAs mediated catalysis (9), among other processes. A single dN and tRNA in human cells, Escherichia coli, Saccha- substitution at random or specific sites in RNA can signif- romyces cerevisiae and, most abundantly, in the RNA icantly affect these processes (5,9–11) by perturbing both genome of dengue virus. The frequency of dNs var- single- and double-stranded RNA structures (5). The incor- ied widely among organisms and sequence contexts, poration of dN into RNA can have a variety of deleteri- and partly reflected the in vitro discrimination ef- ous consequences for cells and RNA viruses. For example, ficiencies of different RNA polymerases against 2- it has been shown that mutation of residues involved in dis- crimination against dNTPs in RNA polymerases can cause deoxyribonucleoside 5 -triphosphates (dNTPs). Fur- growth defects and lethality in cells and viruses (12,13). Fur- ther, we demonstrate a strong link between dN fre- thermore, dNs inserted by cellular and viral RNA poly- quencies in RNA and the balance of dNTPs and ri- merases impede further extension of a transcript (14–16). bonucleoside 5 -triphosphates (rNTPs) in the cellular Beyond the obvious miscoding effects of dN in mRNA, in- pool, with significant stress-induced variation of dN corporation of dN at specific sites in rRNAs and tRNA has incorporation. Potential implications of dNs in RNA been shown to significantly alter critical steps in transla- are discussed, including the possibilities of dN in- tion (10,17–22), including tRNA binding to the ribosome, translocation and elongation. Such alterations may trigger *To whom correspondence should be addressed. Tel: +1 617 253 8017; Fax: +1 617 324 5280; Email: [email protected] Present addresses: Yok Hian Chionh, TyChan, Pte. Ltd., Singapore 068898. Megan E. McBee, TyChan, Pte. Ltd., Singapore 068898. C The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] 2 Nucleic Acids Research, 2016 ribosomal frameshifting (23) or result in the production MATERIALS AND METHODS of prematurely terminated peptides, which has been impli- Cell and virus culture cated in neurodegenerative and other diseases (24). dN in RNA also has implications for replication of viral RNA CCRF-SB human B lymphoblasts (a gift from Dr Jianzhu genomes and reverse-transcription of retroviruses, with in Chen, Singapore-MIT Alliance for Research and Technol- vitro studies revealing problems with initiation and com- ogy) were cultured in RPMI-1640 supplemented with 10% pletion of these two processes when RNA-dependent RNA fetal bovine serum (FBS), 50 units/ml penicillin and 50 ◦ polymerases (RdRPs) and HIV-1 reverse transcriptase were ␮g/ml streptomycin at 37 Cand5%CO2. The cells were presented with dN-containing RNA templates (15,25–29). collected by centrifugation at 350 xg for 10 min at 4◦C. Sac- The presence of dN in viral RNA genomes thus has the po- charomyces cerevisiae strain W1588-4C (a gift from Dr Gra- tential to both alter the rate of viral evolution by mutation ham C. Walker, Massachusetts Institute of Technology) was and cause genome truncation that is associated with persis- grown exponentially in YPDA medium (1% yeast extract, tent viral infections (30). 2% peptone, 2% dextrose, with 0.004% adenine hemisul- In spite of the many potentially deleterious consequences fate) at 30◦C with shaking at 200 rpm. The Δndk mutant of the presence of dN in coding and non-coding RNAs, strain of Escherichia coli W3110 was constructed by trans- this phenomenon is largely unexplored in vivo due to a duction using bacteriophage P1 vir. The mutant gene was lack of appropriate tools for quantitative analysis of dN obtained from the Keio Collection (40) in the form of a gene in RNA. Our understanding of the mechanisms contribut- knockout carrying a kanamycin gene cartridge. The mutant Downloaded from ing to dN incorporation into RNA is based on numerous gene was transduced into E. coli W3110 using a selection in vitro studies of RNA polymerase incorporation of dNs for kanamycin resistance. The resistance cartridge was then during RNA synthesis and the potential for dN incorpo- flipped out using flp sequences surrounding the kanamycin ration in cells with defined nucleotide pool imbalances. In cartridge and flp recombinase supplied from the plasmid general, the concentrations of ribonucleotide triphosphates pCP20 (41). All E. coli strains were grown exponentially http://nar.oxfordjournals.org/ (NTP) range from ∼1000 to ∼3000 ␮M in cells while 2- in LB broth at 37◦C with shaking (220 rpm) to stationary deoxynucleotide triphosphates (dNTP) are ∼10-fold lower phase. The cells were collected by centrifugation (4000 xg in concentration, ranging from ∼100 to ∼300 ␮M (exam- at 4◦C) and washed once with ice-cold PBS. All cells were ples in Table 1)(31–34). Large shifts in these concentra- stored at −80◦C until total RNA extraction. The DENV- tions can occur under a variety of genetic and environmen- 2 strain TSV01 was prepared as described previously (42). tal conditions, such as loss of nucleoside diphosphate kinase Briefly, genome-length viral RNA (vRNA) was in vitro tran- (Ndk) E. coli (Table 1), an enzyme that converts (d)NDPs to scribed from full-length cDNA plasmid linearized by ClaI. their corresponding triphosphates (35). For reasons that are The transcribed RNA is then electroporated into BHK- not entirely clear, loss of ndk disrupts the (d)NTP pool bal- 21 cells, the transfected cells were resuspended in 20 ml of at MIT Libraries on October 6, 2016 ◦ ance to cause large increases in dCTP concentrations (33). DMEM medium and cultured at 30 Cwith5%CO2 for In the context of RNA polymerase activity, the active sites 5 days to produce virus. The virus titer was measured by of most, if not all, RNA polymerases have evolved to ef- plaque assay. To purify virus particles, TSVOI was prop- ficiently exclude dNTPs during RNA synthesis (1). How- agated in mosquito C6/36 cells. Briefly, C6/36 cells were ever, dNTP exclusion is not absolute (1). Kinetic studies infected with TSVOI at a multiplicity of infection (MOI) (14,16,36–38) have revealed that the degree of selectivity for of 0.1 and incubated at 28◦C for 4–5 days. Culture fluids insertion of rNTPs over dNTPs into RNA varies from ∼30- were then harvested, virus particles were precipitated by 8% to ∼5000-fold, depending upon the RNA polymerase and PEG8000 and purified through 10–30% potassium tartrate the rNTP/dNTP pair examined.
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