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.

Recombinant RNA-Dependent RNA Polymerase Complex of Ebola Virus

“…Regardless, the biological relevance of our virus specific observations remains to be investigated; however, it has been reported that RNA viruses are able to incorporate dNTP in their genome. The Dengue virus genome contained on average ~7 DNA residues per genome 35 . Ara-CTP is the triphosphate form of the approved anti-cancer drug cytarabine.…”

Why are Hoogsteen base pairs energetically disfavored in A-RNA compared to B-DNA?

“…Secondly, they provide new insights into how dNMP and rNMP substitutions affect the structures of A-RNA and B-DNA. Such substitutions occur in nature as DNA and RNA polymerases misincorporate rNTPs and dNTPs respectively ( 13 , 14 ). Prior NMR studies indicated that isolated rNMPs in DNA duplexes adopt a C3′-endo sugar pucker ( 73 , 74 ).…”

“…The resulting A-RNA double helix is also more rigid than its B-DNA counterpart ( 7–9 ). These differences between A-RNA and B-DNA have important biological implications for their recognition by proteins ( 10 , 11 ) and ligands ( 12 ), the templated processes of replication, transcription and translation, the consequences of ribonucleotide and deoxyribonucleotide misincorporation ( 13 , 14 ), and the impact of damage ( 15 ) and chemical modifications.…”

Quantifying the RNA cap epitranscriptome reveals novel caps in cellular and viral RNA

“…Total RNA was subsequently extracted with TRIzol following the manufacturer's instructions. The genomic RNA from purified dengue virions was extracted with TRIzol and purified by size-exclusion chromatography as described previously (23,25). The poly(A)-tailed RNA in human CCRF-SB cells was isolated from the total RNA using a Fasttrack MAG Maxi mRNA isolation kit (Life Technologies), whereas the poly(A)-tailed RNA in yeast cells and mouse tissues was isolated from the total RNA using a Dynabeads mRNA Purification kit (Life Technologies) following the manufacturer's protocols.…”