ABSTRACTOverexpressed Angiogenin (ANG) cleaves tRNA anticodons to produce tRNA halves similar to those produced in response to stress, but it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRNA fragments (tRFs) are generated from the tRNA halves. Global short RNAseq experiments reveal that ANG over-expression selectively cleaves a subset of tRNAs (tRNAGlu, tRNAGly, tRNALys, tRNAVal, tRNAHis, tRNAAspand tRNASeC) to produce tRNA halves and 26-30 bases long tRF-5s. Surprisingly, knockout of ANG reveals that the majority of stress-induced tRNA halves except 5’ half from tRNAHisGTGand 3’ half from tRNAAspGTCare ANG-independent, suggesting there are other RNases that can produce tRNA halves. The 17-25 bases long tRF-3s and tRF-5s that could enter into Argonaute complexes are not induced by ANG overexpression, suggesting that they are generated independently from tRNA halves. Consistent with this, knockout of ANG did not decrease tRF-3 levels or gene-silencing activity. Therefore ANG cleaves specific tRNAs, is not the only RNAse that creates tRNA halves and the shorter tRFs are not generated from the tRNA halves or from independent tRNA cleavage by ANG.