Unraveling the molecular mechanism of human polynucleotide phosphorylase (hPNPase) in controlling oxidized RNA

Oxidation by reactive oxygen species is the major source of RNA damaging insult in living organisms. Increased RNA oxidation has been strongly implicated in a wide range of human diseases; predominantly neurodegeneration. Oxidized RNA should be removed from the cellular system to prevent their deleterious effect to the cells and organisms. In eukaryotic cells, mitochondria are the major intracellular sources of ROS and may cause greater damage to the mitochondrial RNA. In this study, we first investigated the RNA oxidation, by measuring the level of 8-hydroxy-Guanosine (8-oxo-Guo), inside mitochondria and cytoplasm in cultured human cells. We discovered that the mitochondrial 8-oxo-Guo is higher than its cytoplasmic counterparts under both normal growth and oxidative stress condition. Next, we explored the role of human polynucleotide phosphorylase (hPNPase) in controlling RNA oxidation inside mitochondria and cytoplasm. hPNPase binds to oxidized RNA with higher affinity, reduces the 8-oxo-Guo level in total RNA and protects cells against oxidative stress. In this study, the molecular mechanism of hPNPase in 8-oxo-Guo reduction was investigated. First, the effect of hPNPase activities on the 8-oxo-Guo level in mitochondria and cytoplasm was examined. The knockdown of hPNPase increased both the mitochondrial and cytoplasmic 8-oxo-Guo, whereas overexpression had the opposite effect. Second, our study revealed that hSUV3, an RNA helicase that forms a functional complex with hPNPase in mitochondria, was dispensable in reducing 8-oxo-Guo levels.