RNA Oxidative Damage and Ribosomal RNA Surveillance under Oxidative Stress

We have studied oxidative damage of RNA, a major type of cellular macromolecules. RNA is a primary target of reactive oxygen species (ROS). Under oxidative stress, most nucleic acid damages in Escherichia coli (E.coli) are present in RNA as shown by the high levels of 8-oxo-G, an oxidized form of guanine. Increased RNA oxidation is closely correlated to cell death under oxidative stress. Surprisingly, neither RNA structure nor association with proteins protects RNA from oxidation. When E. coli cultures were treated with hydrogen peroxide (H2O2), 8-oxo-G forms at higher levels in ribosomal RNA than in non-ribosomal RNA species. The preferential formation of 8-oxo-G in ribosomal RNA is related to the high order structure of the RNA since oxidation produces more 8-oxo-G in native RNA than in denatured RNA, and in a RNA:DNA duplex than in single-stranded RNA of the same sequence. H2O 2-induced 8-oxo-G in ribosomes is removed specifically depending on the activities of polynucleotide phosphorylase (PNPase) and RNase R, two 3' exoribonucleases capable of degrading structured RNA. H2O 2--treatment of E. coli cultures also causes rRNA degradation in a dosage dependent manner. In cells lacking the RNA-degradation exoribonucleases, RNase R, PNPase, and/or RNase II, rRNA fragments accumulated to a high level upon H2O2-treatment. The pattern of the rRNA fragments suggested a specific rRNA degradation pathway that is initiated by endonucleolytic cleavages of 16S and 23S rRNA in the intact ribosomes or subunits of ribosomes, followed by the degradation of the fragments exonucleolytically. Surprisingly, none of the known specific endoribonucleases, RNase E, G, or P, are involved in the initial cleavages of 16S rRNA. Our results demonstrate a major role for RNA degradation in controlling oxidized RNA. We have identified activities that may work in specific pathways for selectively degrading damaged RNA. These activities may play pivotal role in controlling oxidized RNA and protecting cells under oxidative stress.