The cell and stress specific dynamics of in vivo and in vitro canonical and non-canonical tRNA cleavage.

Following stress, tRNA is cleaved to generate tRNA halves (tiRNAs). These stress-induced small RNAs have been shown to regulate translation during stress. To date, angiogenin is considered the main enzyme that cleaves tRNA at its anti-codon site to generate 35 ~ 45 nucleotide long 5' and 3' tiRNA halves, however recent reports indicate the presence of angiogenin-independent cleavage. We previously observed tRNA cleavage pattern occurring away from the anti-codon site. To explore this non-canonical cleavage, we analyze tRNA phenotypical cleavage patterns in rat model of ischemia reperfusion and in two rat cell lines. In vivo mitochondrial tRNAs were prone to this non-canonical cleavage pattern. In vitro, however, both cytosolic and mitochondrial tRNAs could be cleaved non-canonically. We also evaluated the roles of angiogenin and its inhibitor, RNH1, in regulating tRNA cleavage during stress. Our results suggest that mitochondrial stress has an important regulatory role in angiogenin-mediated tRNA cleavage. Angiogenin does not appear to regulate the non-canonical cleavage pattern of tRNA, and RNH1 does not affect it as well. Finally, we verified our previous findings of the stress-specific role of Alkbh1 in regulating tRNA cleavage and showed a strong influence of stress type on Alkbh1-mediated tRNA cleavage and that Alkbh1 impacts non-canonical tRNA cleavage.