Age-driven modulation of tRNA-derived fragments in Drosophila and their potential targets

Abstract

Background: Development of sequencing technologies and supporting computation enable discovery of small RNA molecules that previously escaped detection or were ignored due to low count numbers. While the focus in the analysis of small RNA libraries has been primarily on microRNAs (miRNAs), recent studies have reported findings of fragments of transfer RNAs (tRFs) across a range of organisms.

Results: Here we describe Drosophila melanogaster tRFs, which appear to have a number of structural and functional features similar to those of miRNAs but are less abundant. As is the case with miRNAs, (i) tRFs seem to have distinct isoforms preferentially originating from 5' or 3' end of a precursor molecule (in this case, tRNA), (ii) ends of tRFs appear to contain short "seed" sequences matching conserved regions across 12 Drosophila genomes, preferentially in 3' UTRs but also in introns and exons; (iii) tRFs display specific isoform loading into Ago1 and Ago2 and thus likely function in RISC complexes; (iii) levels of loading in Ago1 and Ago2 differ considerably; and (iv) both tRF expression and loading appear to be age-dependent, indicating potential regulatory changes from young to adult organisms.

Conclusions: We found that Drosophila tRF reads mapped to both nuclear and mitochondrial tRNA genes for all 20 amino acids, while previous studies have usually reported fragments from only a few tRNAs. These tRFs show a number of similarities with miRNAs, including seed sequences. Based on complementarity with conserved Drosophila regions we identified such seed sequences and their possible targets with matches in the 3'UTR regions. Strikingly, the potential target genes of the most abundant tRFs show significant Gene Ontology enrichment in development and neuronal function. The latter suggests that involvement of tRFs in the RNA interfering pathway may play a role in brain activity or brain changes with age.

Fig. 1

Examples of Read Distribution Patterns of tRFs. Screenshot of our RNA display, showing reads that align to tRNA-Ala in the Ago2, 30 days library (a) and MetCAT in Ago1, 30 days (b). Sequence at the bottom with the magenta background indicates single-stranded (loop) regions in the tRNA molecule, while the cyan background and matching grey boxes indicate stems. The red on top indicates read depth coverage of specific regions of the tRNA. Reads (boxes in the middle) with counts of at least 1 % of the most abundant read are displayed; lower count reads are omitted for compact visualization

Fig. 2

Distinct tRF Isoform Changes with Age. Isoform distributions for GluCTC in a Ago1-IP and b Ago2-IP, c AspGTC in Ago1-IP and d Ago2-IP libraries. Both tRFs show a decrease in Ago1, but an increase of specific isoforms in Ago2 with age. Black bars represent normalized counts at 3 days, while red bars represent normalized counts at 30 days. The same GluCTC is also presented in Fig. 3 (GluCTC-2)

Fig. 3

Differential and Preferential Loading. Plot of abundant tRFs that are present in all four libraries. Plots show relative ratios of reads: Ago2 to Ago1 in 3 days (black) and 30 days (red); 30 days to 3 days in Ago1 (slash) and Ago2 (back slash)

Fig. 4

Candidate Seed Regions for tRFs. The numbers of sequence matches in the 3’ UTR regions (a-c) and introns (d-f) are plotted vs window start positions of 7mer windows of (a, d) GlyGCC, (b, e) GlyTCC and (c, f) mt:SerGCT tRFs in Drosophila. Color key for the top row is given in (c), for the bottom row in (f). Expected number of matches is shown in black and average number of matches for all other 7mers with the same nucleotide composition as the given window is shown in blue. The observed number of matches in the D. melanogaster genome is shown in white and in the conserved regions of 12 Drosophila genomes is shown in red

Fig. 5

Share of Seed Alignments by Region. The relative share of seed matches to the most abundant 7mer (as shown in Fig. 4) of (a) GlyGCC and (b) mt:SerAGC

Fig. 6

Examples of Seed Region Matches in Conserved 3’UTRs. Grey highlights and bold text indicate seed complementarity to conserved (12 Drosophila genomes) 3’ UTR regions. Targeted genes with overlapping coordinates in the genome are shown on top. a Both GlyGCC and mir-277 having a 7mer-m8 match, b mt:SerGCT 7mer-m8 match and c GlyGCC having a 7mer-1a match and mt:SerGCT having a 8mer-1a match, with additional A for the 1a matches are also highlighted (mt:SerGCT) or bolded (GlyGCC)