Oxidative stress-mediated tRNA cleavage in HeLa cells.

<p>(A) Minor fraction tiRNAs are generated by longer exposure to 500 µM arsenite (>30 min). The numbers on the left denote the DNA ladder in nt. (B) Arsenite alters the integrity of the 3′-CCA end of full-length tRNAs in a dose-dependent manner. The amount of tRNAs with intact CCA ends was analyzed by their ability to ligate to a fluorescently labeled oligonucleotide (schematic inset) which forms a loop and binds only intact 3′-CCA ends (upper two gels). The intensity of tRNAs with intact CCA termini was quantified from the gels, normalized to the total tRNA amount at each time point and presented as relative values ± SEM (from three independent experiments) to the amount of initial, untreated sample which was set as 1.0. The amount of the total tRNA remained almost unchanged when cells were exposed to arsenite (500 µM) and visualized by SYBR Green (bottom gel marked as total tRNA). (C) Increase of the cellular concentration of angiogenin alters the 3′-CCA integrity of tRNAs. Angiogenin was upregulated by ectopic expression under the control of a CMV promoter for 8 h (+angiogenin). The sample representing arsenite stress (+arsenite) corresponds to the 60-min data point at 500 µM arsenite in panel (B) and is used for comparison. The intensity of tRNAs with intact CCA termini was the quantified as described for panel (B). ** for <i>p</i><0.01.