MicroRNAs (miRNAs) are 18-24 nucleotide (nt) non-coding RNAs that are deeply involved in development, especially in morphogenesis. However, the detailed biological information about 25-45 nt long small RNAs remains unclear. Here, I aimed to perform a comprehensive analysis of 25-45 nt-long small RNAs in Triops cancriformis (Tadpole shrimp), whose morphology dramatically changes during development. I especially focused on stage-specific mitochondrial and genomic tRNA-derived fragments (tRFs).
To detect small RNAs, deep sequencing of small RNA libraries in each six developmental stage (egg, 1st-4tn instar larvae, and adult) of T. cancriformis was performed. In order to analyze tRFs, I obtained mitochondrial DNA sequence from NCBI and genomic DNA sequences by deep sequencing, and predicted mitochondrial and genomic tRNA genes by tRNAscan-SE. By the comparative sequence analysis between tRNA genes and small RNAs, it was found that large amount of small RNAs were derived from mitochondrial and genomic tRNA genes. Interestingly, tRPs were derived from the particular positions of its tRNAs. For example, approximately 25-30 nt-long tRFs were produced from the almost similar positions around the 55 half regions of mitochondrial and genomic tRNALys(CUU) at every six stage. Moreover, expression pattern analysis suggested that the expression of many mitochondrial and genomic tRFs was altered in six developmental stages. Northern blot analysis confirmed that two genomic tRFs were actually expressed in T. cancriformis. In particular, several tRFs in different length were produced from genomic tRNALys(CUU) depending on the developmental stages. These results suggest that tRFs are not random degradation products, but may have important role(s) in T. cancriformis development.
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