(27ad) 8-Oxo-7,8-Dihydroguanosine Alters RNA Degradation Behavior of Polynucleotide Phosphorylase

The impacts of oxidative stress are pervasive across all biomolecules within the cell. Oxidants such as hydrogen peroxide, paraquat, and various air pollutants can lead to the generation of intracellular reactive oxygen species (ROS) that have been shown to oxidize DNA, proteins, lipids, and RNA. The most common oxidized base found in RNA is 8-oxo-7,8-dihydroguanosine (8-oxoG), an RNA modification that has been linked to both mistranslation and reduction in translational activity. Among the few proteins identified that show a binding preference for this RNA modification is polynucleotide phosphorylase (PNPase), an exoribonuclease that represents a constituent of the RNA degradosome. This protein has been shown to be important in cell survival under oxidative stress; however, the molecular mechanisms by which this protein recognizes and acts upon 8-oxoG present in RNA transcripts remains unclear. Here, we demonstrate that PNPase exhibits interesting enzymatic behavior upon encountering 8-oxoG by performing functional biochemical experiments. We further investigate the potential protein regions responsible for this enzyme’s functional behavior and demonstrate that the PNPase catalytic core, rather than the RNA binding domains, are implicated. Finally, we perform additional mutational analysis of regions within the catalytic core based on previous PNPase engineering work to validate the importance of these regions for the activity of the protein in the context of 8-oxoG containing RNA.