Uncovering the Effects of Oxidative RNA Modifications on the Formation of Protein-RNA Complexes

Cells exposed to oxidative stress result in RNA oxidations that are linked to many diseases, including Alzheimer and Parkinson’s diseases. Because RNA oxidations inherently change the chemistry and therefore the configuration of a folded RNA strand, these altered properties can result in changes in RNA-protein interactions. These alterations and their biochemical effects on the fate of RNA molecules are not well understood. To study this gap, we have used as a model E. coli Polynucleotide phosphorylase (PNPase), a RNA-binding protein that interacts with high affinity to 8-oxo-guanosine and is possibly linked to a repair mechanism of oxidized RNA. We have used biochemical methods to study the binding between PNPase and a selected set of RNA oxidations chemistries. We have analyzed the specificity of PNPase for oxidized nucleosides using electrophoretic mobility shift assays (EMSAs). For this, we have cloned the gene encoding E. coli PNPase in an inducible plasmid for recombinant protein expression, transformed it into an E. coli BL21 strain, and induced protein expression by addition of IPTG. After protein purification using the hexahistidine tag in PNPase, EMSAs were performed for determination and quantification of the protein-RNA affinity. We particularly tested the specificity of PNPase binding for the most common types of oxidation occurring in RNA, identified upon oxidative stress of cells exposed to UV, ionizing radiation, and hydrogen peroxide. This approach has resulted in a better understanding of the natural protein recognition mechanisms of PNPase.