Abstract
MicroRNAs (miRNAs) can have organ-specific expression and functions; they can originate from dedicated miRNA genes, from non-canonical miRNA genes, or from mirror-miRNA genes and can also experience post-transcriptional variation. It remains unclear, however, which mechanisms of miRNA production or modification are organ-specific and the extent of their evolutionary conservation. To address these issues, we developed the software Prost! (PRocessing Of Short Transcripts), which, among other features, helps quantify mature miRNAs, accounts for post-transcriptional processing, such as nucleotide editing, and identifies mirror-miRNAs. Here, we applied Prost! to annotate and analyze miRNAs in three-spined stickleback (Gasterosteus aculeatus), a model fish for evolutionary biology reported to have a miRNome larger than most teleost fish. Zebrafish (Danio rerio), a distantly related teleost with a well-known miRNome, served as comparator. Our results provided evidence for the existence of 286 miRNA genes and 382 unique mature miRNAs (excluding mir430 gene duplicates and the vaultRNA-derived mir733), which doesn’t represent a miRNAome larger than other teleost miRNomes. In addition, small RNA sequencing data from brain, heart, testis, and ovary in both stickleback and zebrafish identified suites of mature miRNAs that display organ-specific enrichment, many of which are evolutionarily-conserved in the brain and heart in both species. These data also supported the hypothesis that evolutionarily-conserved, organ-specific mechanisms may regulate post-transcriptional variations in miRNA sequence. In both stickleback and zebrafish, miR2188-5p was edited frequently with similar nucleotide changes in the seed sequence with organ specific editing rates, highest in the brain. In summary, Prost! is a new tool to identify and understand small RNAs, to help clarify a species’ miRNA biology as shown here for an important model for the evolution of developmental mechanisms, and to provide insight into organ-enriched expression and the evolutionary conservation of miRNA post-transcriptional modifications.
Highlights
MicroRNAs are small non-coding RNA molecules about 20-22 nucleotides long that control gene expression post-transcriptionally by repressing translation or inducing the decay of targeted messenger RNA transcripts1–3. miRNAs participate in virtually all biological processes, including the control of cell specification, cell differentiation, organ development, and organ physiology[4,5,6] as well as pathologies in humans and other animals7–10. miRNA genes appear to be evolutionarily-conserved in number, sequence, and www.nature.com/scientificreports/
Other pathways and other gene types can produce miRNAs (e.g. miRNAs from Drosha- or Dicer-independent pathways, miRNAs produced by both DNA strands at the same locus, lncRNAs, and snoRNAs18–21) and the most common alternative miRNA biogenesis pathway is the processing of miRtrons, which are miRNA hairpins originating from spliced introns of protein-coding genes[22,23]
Length modifications at the 5′ end of miRNAs occur less frequently than at the 3′ end, perhaps because they cause a shift in the seed, which can modify the identity of targeted transcripts and can drastically change the miRNA’s function26,27. miRNA sequence variation can occur due to post-transcriptional editing, in which ADAR enzymes post-transcriptionally modify a nucleotide, usually an adenosine (A), into another base, usually an inosine (I)[28,29,30]
Summary
MicroRNAs (miRNAs) are small non-coding RNA molecules about 20-22 nucleotides long that control gene expression post-transcriptionally by repressing translation or inducing the decay of targeted messenger RNA transcripts (mRNAs)1–3. miRNAs participate in virtually all biological processes, including the control of cell specification, cell differentiation, organ development, and organ physiology[4,5,6] as well as pathologies in humans and other animals7–10. miRNA genes appear to be evolutionarily-conserved in number, sequence, and www.nature.com/scientificreports/. MiRNA sequence variation can occur due to post-transcriptional editing, in which ADAR (adenosine deaminase, RNA-specific) enzymes post-transcriptionally modify a nucleotide, usually an adenosine (A), into another base, usually an inosine (I)[28,29,30]. Other tools make use of genomic alignments and specialize in the discovery of novel miRNAs36,43 or the study of isomiRs35,44 These tools often perform well for their respective functions, but in many cases, lack transparency in their filtering and annotating algorithms, have few user-defined parameter choices that might help tune a user’s specific application, and/or lack the ability to inspect the entire small RNA dataset and omit sequences not already annotated as a miRNA. While many tools are available to study small RNA sequencing datasets, current tools usually do not provide a comprehensive, genome-based analysis of small RNA datasets, limiting the study of the full complexity of an experiment by failing to report some of the post-transcriptional processes affecting the diversity of small RNAs
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