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 103 to 105 ribonucleotide (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: Jin Wang, Hongping Dong, Yok Hian Chionh, Megan E McBee, Sasilada Sirirungruang, Richard P Cunningham, Pei-Yong Shi, Peter C Dedon
• Journal: Nucleic acids research (Nucleic Acids Res) Vol. 44 Issue 18 Pg. 8962-8975 (Oct 14 2016) ISSN: 1362-4962 [Electronic] England
• PMID: 27365049 (Publication Type: Journal Article)
• Copyright: © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Chemical References

• Deoxyribonucleotides
• RNA, Bacterial
• RNA, Viral
• Ribonucleotides
• RNA

Topics

• Animals
• Base Composition
• Cell Line
• Chromatography, Liquid
• Deoxyribonucleotides (chemistry)
• Eukaryotic Cells (metabolism)
• Humans
• Hydrolysis
• Mammals
• Mutagenesis
• Prokaryotic Cells (metabolism)
• RNA (chemistry, genetics)
• RNA, Bacterial (chemistry, genetics)
• RNA, Viral (chemistry, genetics)
• Ribonucleotides
• Stress, Physiological (genetics)
• Tandem Mass Spectrometry