Abstract
tRNA fragments (tRFs) and tRNA halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more. Overexpressed angiogenin (ANG) cleaves tRNA anticodons and produces tRNA halves similar to those produced in response to stress. However, it is not clear whether endogenous ANG is essential for producing the stress-induced tRNA halves. It is also not clear whether smaller tRFs are generated from the tRNA halves. Here, using global short RNA-Seq approach, we found that ANG overexpression selectively cleaves a subset of tRNAs, including tRNAGlu, tRNAGly, tRNALys, tRNAVal, tRNAHis, tRNAAsp, and tRNASeC to produce tRNA halves and tRF-5s that are 26-30 bases long. Surprisingly, ANG knockout revealed that the majority of stress-induced tRNA halves, except for the 5' half from tRNAHisGTG and the 3' half from tRNAAspGTC, are ANG independent, suggesting there are other RNases that produce tRNA halves. We also found that 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, ANG knockout did not decrease tRF-3 levels or gene-silencing activity. We conclude that ANG cleaves specific tRNAs and is not the only RNase that creates tRNA halves and that the shorter tRFs are not generated from the tRNA halves or from independent tRNA cleavage by ANG.
Highlights
TRNA fragments and tRNA halves have been implicated in various cellular processes, including gene silencing, translation, stress granule assembly, cell differentiation, retrotransposon activity, symbiosis, apoptosis, and more
The two halves produced by tRNA cleavage are named respectively as 5Ј tRNA halves and 3Ј tRNA halves. 5Ј tRNA halves have been shown to inhibit global translation and promote stress granule assembly [18, 19], whereas 3Ј tRNA halves interact with cytochrome c and protect cells from stress-induced apoptosis [20]. (c) Similar to 3Ј tRNA halves, tRNA fragments (tRFs)-3s end at the 3Ј end of mature tRNAs, but start by cleavage within the T-loop
This is consistent with a previous report showing some specificity of recombinant ANG toward certain tRNAs by a customized tRF microarray approach [45] and another report showing that ANG does not produce halves from tRNATyr [16]
Summary
To identify which tRNAs are cleaved and how by ANG, we overexpressed full-length human ANG in HEK293T cells (Fig. 1B and Fig. S1A). This size range will capture tRNA fragments but not the full-length tRNAs (Fig. S1D). We define 3Ј fragments irrespective of CCA presence to account for potential cleavage of the 3Ј CCA at CA dinucleotide by ANG [42] Long tRF-5s of 26 –30 nt ( called td-piRNAs) were increased by ANG overexpression (Fig. 1D), it is not clear whether they are directly created by cleavage in the anticodon stem or are further processed from the 3Ј end of the 5Ј tRNA halves
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