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. 2015 Jul 14:16:83.
doi: 10.1186/s12863-015-0245-5.

Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp)

Yuka Hirose  1   2 , Kahori T Ikeda  3   4 , Emiko Noro  5 , Kiriko Hiraoka  6 , Masaru Tomita  7   8   9 , Akio Kanai  10   11   12
Affiliations

Precise mapping and dynamics of tRNA-derived fragments (tRFs) in the development of Triops cancriformis (tadpole shrimp)

Yuka Hirose et al. BMC Genet. .

Abstract

Background: In a deep sequencing analysis of small RNAs prepared from a living fossil, the tadpole shrimp Triops cancriformis, a 32-nt small RNA was specifically detected in the adult stage. A nucleotide sequence comparison between the 32-nt small RNA and predicted tRNA sequences in the draft nuclear genomic DNA showed that the small RNA was derived from tRNA(Gly)(GCC). To determine the overall features of the tRNA-derived fragments (tRFs) of T. cancriformis, the small RNA sequences in each of the six developmental stages (egg, 1st-4th instar larvae, and adult) were compared with the mitochondrial and nuclear tRNA sequences.

Results: We found that the tRFs were derived from mitochondrial and nuclear tRNAs corresponding to 16 and 39 anticodons, respectively. The total read number of nuclear tRFs was approximately 400 times larger than the number of mitochondrial tRFs. Interestingly, the main regions in each parental tRNA from which these tRFs were derived differed, depending on the parental anticodon. Mitochondrial tRF(Ser)(GCU)s were abundantly produced from the 5' half regions of the parental tRNA, whereas mitochondrial tRF(Val)(UAC)s were mainly produced from the 3' end regions. Highly abundant nuclear tRFs, tRF(Gly)(GCC)s, tRF(Gly)(CCC)s, tRF(Glu)(CUC)s, and tRF(Lys)(CUU)s were derived from the 5' half regions of the parental tRNAs. Further analysis of the tRF read counts in the individual developmental stages suggested that the expression of mitochondrial and nuclear tRFs differed during the six stages. Based on these data, we precisely summarized the positions of the tRFs in their parental tRNAs and their expression changes during development.

Conclusions: Our results reveal the entire dynamics of the tRFs from both the nuclear and mitochondrial genomes of T. cancriformis and indicate that the majority of tRFs in the cell are derived from nuclear tRNAs. This study provides the first examples of developmentally expressed mitochondrial tRFs.

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Figures

Fig. 1
Fig. 1
Pie charts summarizing the proportions of mitochondrial and nuclear tRFs in T. cancriformis. The tRFs were categorized by their corresponding anticodons
Fig. 2
Fig. 2
Four examples of mitochondrial tRNAs and their tRFs in T. cancriformis. Secondary structures of four mature mitochondrial tRNAs are shown on the left. Nucleotide sequence alignments of each tRNA and its tRFs are shown in the center. Total read counts of each tRF in the six developmental stages are shown (the three most numerous of the tRF variants are indicated with arrows and Roman numerals i–iii). On the right, the main tRF regions in each parental tRNA are shown with a bold line. AC stem–loop region (anticodon stem–loop region). See Additional file 1: Figure S4 for another 12 examples of mitochondrial tRNAs and their tRFs in T. cancriformi
Fig. 3
Fig. 3
Expression of four mitochondrial tRFs during T. cancriformis development. (a) Heatmap shows the expression profiles of mitochondrial tRFs during T. cancriformis development. Each color in the heatmap represents the relative normalized read counts of the three most-enriched tRFs. Red indicates a high relative read frequency and green indicates a low relative read frequency. (E) Egg; (1) 1st instar larva; (2) 2nd instar larva; (3) 3rd instar larva; (4) 4th instar larva; (a) adult. Bar graph shows the total normalized read counts (in all stages) of the three most-enriched tRFs among the tRF variants. (b) Accumulation of all tRF reads that mapped to four individual mature mitochondrial tRNA sequences are visualized in each of the six developmental stages (also see Fig. 2 and Additional file 1: Figure S5). Vertical axis indicates the sum of the normalized read counts, and the horizontal axis indicates the base position of each tRNA from the 5’ to 3’ end. “E”, “1–4”, and “A” indicate egg, 1st–4th instar larvae, and adult, respectively
Fig. 4
Fig. 4
Four examples of nuclear tRNAs and their tRFs in T. cancriformis. Examples of highly abundant nuclear tRFs were selected. The secondary structures of the four mature nuclear tRNAs and the nucleotide sequence alignments between these tRNAs and their tRFs are shown (also see Fig. 2 legend). Different tRNA genes but with the same anticodon sequences (tRNA gene subtypes) are shown with upper-case Roman characters (I–V). The white circle in the secondary structure indicates the nonconserved nucleotides among the tRNA subtypes. Highly redundant tRF reads (≥500 reads) were used for the sequence alignments. Also see Additional file 1: Figure S6 for another 12 examples of nuclear tRNAs and their tRFs in T. cancriformis
Fig. 5
Fig. 5
Expression of four nuclear tRFs during T. cancriformis development. The accumulation of all tRF reads that mapped to four individual mature nuclear tRNA sequences are visualized in each of the six developmental stages, as in Fig. 3B (also see Fig. 4 and Additional file 1: Figure S7)
Fig. 6
Fig. 6
Northern blot analysis of two T. cancriformis nuclear tRFs. Expression of (a) nuclear tRFGly(GCC) and (b) nuclear tRFLys(CUU) was detected in the adult stage of T. cancriformis with a northern blotting analysis using probes specific to the most-enriched tRF for each anticodon. Total RNA (1 or 4 μg) isolated from the adult stage was used. Washing treatments were performed at either 45 or 55 °C
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