All arsenic compounds elicit a number of responses including growth inhibition, induction of DNA strand breaks and the production of reactive oxygen species, indicating toxicity, which can result in cell death ( 17, 18). A particular stressor causing robust tRNA fragmentation in eukaryotic cells is inorganic sodium arsenite, NaAsO 2 (As). For instance, heat shock, oxidative stress, methionine or nitrogen starvation, and stationary phase conditions caused detectable tRNA fragmentation in yeast, while glucose or general amino acid starvation as well as UV exposure did not ( 5). Of note, some stressors result in tRNA fragmentation while others do not. Upon stress, ANG phosphorylation causes the dissociation from its inhibitor RNH1 ( 13) and the activation of its catalytic activity results in targeting of pyrimidine-purine dinucleotide sequences, preferentially in the loop structures of tRNAs ( 6, 14–16). Angiogenin (ANG), a member of the RNase A family, is the main nuclease of various redundant nucleases capable of tRNA hydrolysis ( 6, 8, 12). Stress-induced tsRNAs are the result of tRNA hydrolysis in the anticodon loop, which is performed by members of two nuclease families (RNase A and RNase T2). Specifically, the production of tsRNAs in the form of tRNA halves has been reported after starvation ( 4), oxidative stress ( 5, 6), nutritional deficiency ( 7), hypoxia and hypothermia ( 8, 9), heat shock and gamma-irradiation ( 6, 10, 11). tsRNAs have been detected in almost every cellular context, during various developmental stages and, importantly, during exposure to defined stress conditions ( 3). An increasing body of work has assigned functional relevance to various tsRNAs because their occurrence correlated not only with cellular stress responses but also with complex molecular and cellular processes including immunity, cancer, neurodegeneration and the intergenerational inheritance of information ( 1, 2). The biogenesis of tRNA-derived small RNAs (tsRNAs), their biological impact and their potential as biomarkers have been the subject of intense scrutiny in recent years. These findings suggest a need to modify current experimental stress paradigms in order to allow separating the function of tRNA fragmentation during the acute stress response from tRNA fragmentation as a consequence of ongoing cell death, which will have major implications for the current perception of the biological function of stress-induced tsRNAs. Furthermore, the increased presence of tsRNA species in culture medium collected from stressed cells indicated that cells suffering from experimental stress exposure gave rise to stable extracellular tsRNAs. Quantification of specific tsRNA species in cells responding to experimental stress and in cells that were transfected with synthetic tsRNAs indicated that neither physiological nor non-physiological copy numbers of tsRNAs induced the formation of stress granules. These experiments revealed that exposure to stress parameters commonly used to induce tRNA fragmentation negatively affected cell viability after stress removal. Various cell culture models were exposed to oxidative stressors followed by determining cell viability, the production of specific tsRNAs and stress granule formation.
Here, we have revisited accepted experimental paradigms for modeling oxidative stress resulting in tRNA fragmentation. tRNA-derived small RNAs (tsRNAs) have been associated with many cellular processes, including improved survival during stress conditions. TRNA fragmentation is an evolutionarily conserved molecular phenomenon.
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