Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation.

Abstract

RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect <i>E<i>scherichia</i> coli</i> cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of <i>E. coli</i> PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through <i>E. coli</i> cell growth experiments under H₂O₂-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress.