Comprehensive annotation of tRNAs and Characterization of tRNA-derived fragments in planaria

Freshwater planarians are well known for their immense regenerative capacity. Regeneration in this organism is systematically coordinated by gene regulatory programs, which includes post-transcriptional regulation. Small RNAs have been central to many post-transcriptional gene regulatory processes and recently small RNAs derived from tRNAs have been implicated in the regulation of various biological processes such as stem cell differentiation, paternal inheritance and in cell stress response. Previous studies on planarian small RNAs failed to report tRNA fragments mainly due to the absence of a comprehensive tRNA annotation. In this study, we have confidently identified and annotated 457 tRNA loci in S.mediterranea using two tRNA prediction programs, tRNAScan-SE and Aragorn. Our annotation of tRNAs facilitated identification of tRNA fragments in planarians. Bioinformatics analysis revealed three main species of tRNA fragments: the 18 to 24 nt tRF5s and itRFs, and the abundant 32 nt 5tsRNAs. Profiling of tRNA derived fragments in the transverse sections of the planaria spanning the whole animal revealed diverse expression patterns of these small RNAs, including those that are enriched in the anterior region towards the head and the pharynx. Expression analysis of the three different tRNA fragment species revealed dynamic expression of these small RNAs over the course of planarian regeneration. Our study also identified tRNA fragments that are upregulated either specifically during early or late regeneration. Finally, our analysis of the tRNA fragments revealed that the 18 to 24 nt itRFs could be potentially processed by DICER whereas the 25 to 30 nt 5tsRNAs were found associated with PIWI proteins.