Figure 5.

Impact of Alkbh1 overexpression on different cytosolic and mitochondrial tRNA species’ cleavage show stress and tRNA specific patterns. A: Cytosolic tRNA cleavage in B35^Mock and B35^ALK cells after 4 hours of As (1 mM) or A.M (200 µg/ml) stresses. Northern blotting performed with 1 µg total RNA per lane, Alkbh1 overexpression led to a general pattern of increased tRNA cleavage. Leu^AAG, Ala^TGC, Gly^GCC, Glu^CTC, Lys^CTT and Arg^CCT showed increased cleavage in their 5ʹtiRNA fragments (whether canonical or larger fragments) in a pattern opposite to what was observed with Alkbh1 KD (Fig. 3). Moreover, Met^CAT and Gln^CTG, which did not exhibit apparent cleavage with stress and siRNA KD, were cleaved with Alkbh1 overexpression into large 5ʹtiRNA fragments after A.M stress (red arrows). 5 s rRNA is provided as a loading control. B: Mitochondrial tRNA cleavage in B35^Mock and B35^ALK cells after As and A.M stresses. Northern blotting performed with 4 µg total RNA per lane. mt-Tyr^GTA cleavage after As in B35^ALK cells was not impacted in an apparent manner, however, after A.M stress, the larger fragments (red arrow), which were reduced after siRNA KD in Fig. 3, did exhibit some increase in its signal. mt-Met^CAT cleavage after A.M stress in B35^ALK cells was greatly enhanced, with the larger fragment (red arrow) being clearly visible and the appearance of 2 other fragments that were not observable prior to Alkbh1 overexpression (black arrows). mt-Asn^GTT showed an enhanced cleavage after Alkbh1 overexpression (red arrow), with the observation of faint signal of a smaller 5ʹtiRNA fragment not observable prior to overexpression (black arrow). mt-Gly^TCC, mt-Arg^TCG and mt-Ala^TGC that were not cleaved prior to Alkbh1 overexpression showed no impact of Alkbh1 on their levels.