Small RNA sequencing of extra-cellular RNAs from arsenite-stressed HeLa cells and mouse embryonic fibroblasts

Stress-induced tRNA fragmentation is an evolutionarily conserved molecular phenomenon. A variety of the resulting tRNA-derived small RNAs (tsRNAs) have been associated with a multitude of cellular processes including cell survival during adverse environmental conditions. However, application of experimental stressors under laboratory conditions often differs from stress encountered naturally. This raises important questions about the extent of experimental bias when studying stress biology including tRNA fragmentation, especially in cell culture. Here, we have revisited one of the most often used experimental paradigms for modeling oxidative stress and tRNA fragmentation, the exposure of cultured cells to sodium arsenite. These experiments revealed that transient exposure to sodium arsenite concentrations that caused robust tRNA fragmentation resulted in extensive cell death during the stress recovery. Importantly, released material from dying cells contained also tsRNA species, which were sufficiently stable against nuclease digestion. Overall design: In order to determine the identity of RNAs that were sufficiently stable for 24 hours after being released by dying cells that had been transiently exposed to sodium arsenite (0.75 mM for one hour), cell culture medium was collected after 24 hours recovery, all RNAs were extracted and sequenced.