BackgroundDevelopment 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.ResultsHere 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.ConclusionsWe 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.ReviewersThis article was reviewed by Eugene Koonin, Neil Smalheiser and Alexander Kel.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-015-0081-6) contains supplementary material, which is available to authorized users.

Transfer RNA fragments (tRFs) are an emerging class of small RNA molecules derived from mature or precursor tRNAs. They are found across a wide range of organisms and tissues, in small RNA fraction or loaded to Argonaute in numbers comparable to microRNAs. Their functions and mechanisms of action are largely unknown, and results obtained on individual tRFs are often hard to generalize. Here we predicted binding mechanisms and specific target interaction sites of 26 human Argonaute-loaded tRFs of different types using large-scale meta-analyses of available experimental data. Strikingly, our findings matched all interaction sites detected in a recent experimental screen, confirming the validity of our computational approach. Such sites are primarily located on the 5ʹ end of tRFs and often involve additional binding along the tRF length, similar to microRNAs. Indicative of multiple layers of regulation, diverse regulatory non-coding RNAs comprised a third of the tRF targets, with the rest being proteincoding transcripts. In the latter, coding sequence and 3ʹ UTRs were the likely primary target regions, although we observed interactions of tRFs with 5ʹ UTRs. Another novel phenomenon we report, a large number of putative interactions between tRFs and introns, is compatible with the roles of Argonaute in the nucleus. Further, observed tRF-intron binding modes suggest a mechanism of interaction of tRFs with Argonaute-dependent introns, and we predict here >20 candidate introns of this type. Taken together, these results present tRFs as regulatory molecules with a rich functional spectrum.

Background: The progress of next-generation sequencing technologies has unveiled various non-coding RNAs that have previously been considered products of random degradation and attracted only minimal interest. Among small RNA families, microRNA (miRNAs) have traditionally been considered key post-transcriptional regulators. However, recent studies have reported evidence for widespread presence of fragments of tRNA molecules (tRFs) across a range of organisms and tissues, and of tRF involvement in Argonaute complexes.  Methods:To elucidate potential tRF functionality, we compared available RNA sequencing datasets derived from the brains of young, mid-aged and old rats. Using sliding 7-mer windows along a tRF, we searched for putative seed sequences with high numbers of conserved complementary sites within 3' UTRs of 23 vertebrate genomes. We analyzed Gene Ontology term enrichment of predicted tRF targets and compared their transcript levels with targets of miRNAs in the context of age.  Results and Discussion: We detected tRFs originating from 3’- and 5’-ends of tRNAs in rat brains at significant levels. These fragments showed dynamic changes: 3’ tRFs monotonously increased with age, while 5’ tRFs displayed less consistent patterns. Furthermore, 3’ tRFs showed a narrow size range compared to 5’ tRFs, suggesting a difference in their biogenesis mechanisms. Similar to our earlier results in Drosophila and compatible with other experimental findings, we found “seed” sequence locations on both ends of different tRFs. Putative targets of these fragments were found to be enriched in neuronal and developmental functions. Comparison of tRFs and miRNAs increasing in abundance with age revealed small, but distinct changes in brain target transcript levels for these two types of small RNA, with the higher proportion of tRF targets decreasing with age. We also illustrated the utility of tRF analysis for annotating tRNA genes in sequenced genomes.

Comparative genomics studies typically limit their focus to single nucleotide variants (SNVs) and that was the case for previous comparisons of woolly mammoth genomes. We extended the analysis to systematically identify not only SNVs but also larger structural variants (SVs) and indels and found multiple mammoth-specific deletions and duplications affecting exons or even complete genes. The most prominent SV found was an amplification of RNase L (with different copy numbers in different mammoth genomes, up to 9-fold), involved in antiviral defense and inflammasome function. This amplification was accompanied by mutations affecting several domains of the protein including the active site and produced different sets of RNase L paralogs in four mammoth genomes likely contributing to adaptations to environmental threats. In addition to immunity and defense, we found many other unique genetic changes in woolly mammoths that suggest adaptations to life in harsh Arctic conditions, including variants involving lipid metabolism, circadian rhythms, and skeletal and body features. Together, these variants paint a complex picture of evolution of the mammoth species and may be relevant in the studies of their population history and extinction.

Ancient DNA (aDNA) studies often rely on standard methods of mutation calling, optimized for high-quality contemporary DNA but not for excessive contamination, time- or environment-related damage of aDNA. In the absence of validated datasets and despite showing extreme sensitivity to aDNA quality, these methods have been used in many published studies, sometimes with additions of arbitrary filters or modifications, designed to overcome aDNA degradation and contamination problems. The general lack of best practices for aDNA mutation calling may lead to inaccurate results. To address these problems, we present ARIADNA (ARtificial Intelligence for Ancient DNA), a novel approach based on machine learning techniques, using specific aDNA characteristics as features to yield improved mutation calls. In our comparisons of variant callers across several ancient genomes, ARIADNA consistently detected higher-quality genome variants with fast runtimes, while reducing the false positive rate compared with other approaches.