Oxidized messenger RNA induces translation errors

Abstract

To investigate the effect of RNA oxidation on normal cellular functions, we studied the translation of nonoxidized and oxidized luciferase mRNA in both rabbit reticulocyte lysate and human HEK293 cells. When HEK293 cells transfected with nonoxidized mRNA encoding the firefly luciferase protein were cultured in the presence of paraquat, there was a paraquat concentration-dependent increase in the formation of luciferase short polypeptides (SPs) concomitant with an increase in 8-oxoguanosine. Short polypeptides were also formed when the mRNA was oxidized in vitro by the Fe-ascorbate-H2O2 metal-catalyzed oxidation system before its transfection into cells. Translation of the in vitro oxidized mRNA in rabbit reticulocyte lysate also led to formation of SPs. The SPs formed by either procedure contained the N-terminal and the C-terminal portions of the tagged luciferase. In addition, the oxidized mRNA was able to associate with ribosomes to form polysomes similar to those formed with nonoxidized mRNA preparations, indicating that the oxidized mRNAs are mostly intact; however, their translation fidelity was significantly reduced. Nevertheless, our results indicate that the SPs were derived from both premature termination of the translation process of the oxidized mRNA and the proteolytic degradation of the modified full-length luciferase resulting from translation errors induced by oxidized mRNA. In light of these findings, the physiological consequences of mRNA oxidation are discussed.8-oxoguanosine ͉ firefly luciferase ͉ premature termination ͉ RNA oxidation ͉ reactive oxygen species T he generation of reactive oxygen species (ROS) is an unavoidable cellular event in normal respiring cells. ROS are known to play an important role in normal physiological functions and they can also oxidize cellular macromolecules, such as nucleic acids, proteins, and lipids, and lead to the loss of their physiological functions (1, 2). Growing evidence indicates that increased oxidative damage appears to be associated with a wide range of age-related neurodegenerative conditions. Although oxidation of DNA may ultimately have deleterious consequences on cellular homeostasis, multiple DNA repair systems are available to minimize such damage (3). Because of the fact that RNAs are less protected against ROS under oxidative stress conditions, intracellular RNA was found to be more susceptible to oxidation than DNA (4, 5). Similarly, irradiation of human skin fibroblasts with 765-kJ/m 2 UV A induced more extensive RNA oxidation than DNA oxidation (6). The oxidation of rRNA, tRNA, and mRNA may have serious effects on cellular homeostasis, because oxidation of these RNAs could impair the overall integrity of translational processes. Moreover, it was recently shown that oxidation of RNA is implicated in several neurodegenerative diseases (7-9). Nunomura et al. (8) reported that cytoplasmic RNA oxidation occurs to a greater extent in neurons that are implicated in the development of Alzheimer's disease (AD). Shan et al. (10) found that signif...

Ammonia induces RNA oxidation in cultured astrocytes and brain in vivo†

“…13 Messenger RNA oxidation is associated with a decreased translation efficacy even in intact ribosomes and the production of defective proteins caused by impaired translation fidelity. 16,35 Oxidation of astroglial GLAST mRNA by ammonia (Figs. 4, 8) may explain in part the known decrease of GLAST expression and glutamate uptake in cultured astrocytes, 36 which contributes to ammonia-induced excitotoxicity.…”

Nucleic acid oxidation in Alzheimer disease

“…Oxidized mRNAs lead to loss of normal protein level and protein function and potentially produce defective proteins leading to protein aggregation, a common feature of neurodegenerative diseases [150]. Recently it has been shown that the translation of oxidized mRNA in cell lines causes the accumulation of short polypeptides, which is a result of premature termination of the translation process of the oxidized mRNA and/ or the proteolytic degradation of the modified protein containing the translation errors due to the oxidized mRNA [151]. Additionally, it has also been shown that the oxidative damage to E. coli 16S rRNA results in the formation of short complementary DNA (cDNA) [152].…”

Emerging Roles of the Nucleolus in Regulating the DNA Damage Response: The Noncanonical DNA Repair Enzyme APE1/Ref-1 as a Paradigmatical Example

“…While oxidative damage to DNA is essentially repaired through the base excision repair (BER) pathway, no evidences of similar repair processes have been described for RNA molecules, even though some BER proteins recently entered the arena of the RNome world (138). If not repaired, damage to RNA molecules may lead to ribosomal dysfunctions and erroneous translation; thus, significantly affecting the overall protein synthesis mechanism (38,135). Moreover, RNA damage has been shown to cause cell cycle arrest and cell death with or without the contribution of p53 and inhibition of protein synthesis (11).…”