The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to f... More
The oxidation of RNA has been implicated in the development of many diseases. Among the four ribonucleotides, guanosine is the most susceptible to oxidation, resulting in the formation of 8-oxo-7,8-dihydroguanosine (8-oxoG). Despite the limited knowledge about how cells regulate the detrimental effects of oxidized RNA, cellular factors involved in its regulation have begun to be identified. One of these factors is polynucleotide phosphorylase (PNPase), a multifunctional enzyme implicated in RNA turnover. In the present study, we have examined the interaction of PNPase with 8-oxoG in atomic detail to provide insights into the mechanism of 8-oxoG discrimination. We hypothesized that PNPase subunits cooperate to form a binding site using the dynamic SFF loop within the central channel of the PNPase homotrimer. We evolved this site using a novel approach that initially screened mutants from a library of beneficial mutations and assessed their interactions using multi-nanosecond Molecular Dynamics simulations. We found that evolving this single site resulted in a fold change increase in 8-oxoG affinity between 1.2 and 1.5 and/or selectivity between 1.5 and 1.9. In addition to the improvement in 8-oxoG binding, complementation of K12 Δpnp with plasmids expressing mutant PNPases caused increased cell tolerance to H2O2. This observation provides a clear link between molecular discrimination of RNA oxidation and cell survival. Moreover, this study provides a framework for the manipulation of modified-RNA protein readers, which has potential application in synthetic biology and epitranscriptomics.,© 2019 The Authors.
