Experimental paradigms revisited: oxidative stress-induced tRNA fragmentation does not correlate with stress granule formation but is associated with delayed cell death

Abstract

tRNA fragmentation is an evolutionarily conserved molecular phenomenon. tRNA-derived small RNAs (tsRNAs) have been associated with many cellular processes, including improved survival during stress conditions. Here, we have revisited accepted experimental paradigms for modeling oxidative stress resulting in tRNA fragmentation. Various cell culture models were exposed to oxidative stressors followed by determining cell viability, the production of specific tsRNAs and stress granule formation. These experiments revealed that exposure to stress parameters commonly used to induce tRNA fragmentation negatively affected cell viability after stress removal. 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. 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. 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.

Transfer RNAs-derived small RNAs and their application potential in multiple diseases

“…Such non-canonical cleavage takes place in vivo mainly in mitochondrial tRNAs, which in vitro also produced multiple large-sized tiRNAs depending on the cutting sites yet was not limited to mitochondrial but also cytoplasmic tRNA transcripts (Rashad et al, 2021). Sanadgol et al (2022) recently confirmed that medium collected from cells growing in stable conditions contained various ex-tsRNAs which were just slightly shorter than arsenite-induced tsRNAs, indicating tsRNAs that represented the acute phase of cells should be distinguished from ex-tsRNAs, because the latter might be derived from Frontiers in Cell and Developmental Biology frontiersin.org cellular repair processes (Sanadgol et al, 2022). Further studies about the biogenesis of tsRNAs are needed to bring new insights into this new category of noncoding RNAs.…”

“…However, the accepted notion that tiRNAs are beneficial to cell survival under stress conditions is challenged recently. Sanadgol et al (2022) had newly discovered a tRNA-Gly-derived tiRNA which could be induced by arsenite promoted the cell death suggesting the diverse functions of tiRNAs in cell viability ( Sanadgol et al, 2022 ).tiRNAs, as novel non-coding RNAs, are garnering increasing focus in the field of study, but their functions and mechanisms remain largely unknown.…”

Stress Granule Homeostasis, Aberrant Phase Transition, and Amyotrophic Lateral Sclerosis

“…Therefore, the true role of tiRNA-associated SG formation in human disease needs to be further explored. 53 The regulation of SG homeostasis also involves the methylation of mRNAs. In vitro assays indicated that poly N6-methyladenosine (poly m 6 A) modified RNA strand can play as a scaffold to promote the LLPS of the m 6 A binding proteins: YTHDFs.…”

Nicked tRNAs are stable reservoirs of tRNA halves in cells and biofluids

“…Knock-out of RNase 1 in K562 cells also shaped exRNA profiles from tRNA halves into full-length tRNAs (Nechooshtan et al, 2020). Thus, most nonvesicular tRNA halves are generated directly in the extracellular space by endonucleolytic cleavage of extracellular full-length tRNAs (Nechooshtan et al, 2020; Sanadgol et al, 2022; Tosar et al, 2020).…”