Abstract

Numerous studies on microRNAs (miRNA) in cancer and other diseases have been accompanied by diverse computational approaches and experimental methods to predict and validate miRNA biological and clinical significance as easily accessible disease biomarkers. In recent years, the application of the next-generation deep sequencing for the analysis and discovery of novel RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected by (patho)physiological changes, population origin, individual’s gender, and age. Here, we provide an in-depth overview of currently available bioinformatics approaches for the detection and visualization of both mature miRNA and cognate isomiR sequences. An attempt has been made to present in a systematic way the advantages and downsides of in silico approaches in terms of their sensitivity and accuracy performance, as well as used methods, workflows, and processing steps, and end output dataset overlapping issues. The focus is given to the challenges and pitfalls of isomiR expression analysis. Specifically, we address the availability of tools enabling research without extensive bioinformatics background to explore this fascinating corner of the small RNAome universe that may facilitate the discovery of new and more reliable disease biomarkers.

Highlights

  • RNA biomarkers has clearly shown an expanding repertoire of diverse sequence variants of mature miRNAs, or isomiRs, resulting from alternative post-transcriptional processing events, and affected byphysiological changes, population origin, individual’s gender, and age

  • The encoding loci of some miRNAs reside in introns or untranslated regions of protein-coding genes, as well as in introns or exons of non-coding RNAs [6,7]

  • The entire pre-miRNA instead of binding to Dicer is loaded on AGO2, which is the only one of all AGO proteins that has a slicer activity, and the second cleavage step of the 3p strand is performed followed by 30 -50 trimming to complete the miR-451 maturation [28,29]

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Summary

Canonical miRNA Biogenesis Pathway

MIR genes are transcribed in a manner similar to protein-coding genes by RNA polymerase-II (Pol II), and occasionally by RNA polymerase III (Pol III) to yield primary miRNAs (pri-miRNAs) and undergo multistep processing [9,10]. Each pri-miRNA forms a hairpin-shaped structure which is processed further by a microprocessor complex, consisting of one molecule of the ribonuclease III (RNase III). DGCR8 directs Drosha to the double stranded RNA (dsRNA)–single stranded RNA (ssRNA) junction on the pri-miRNAs to cleave 11 bp away from the junction releasing. The RNase III enzyme Dicer, together with the dsRNA-binding proteins TRBP (transactivation response element RNA-binding protein) and/or PACT (protein activator of the interferon-induced protein kinase), further cleaves pre-miRNA to generate a short double-stranded RNA fragment [15,16,17]. AGO and the retained ssRNA form the mature miRNAinduced silencing complex (miRISC). It was reported that the half-lives of miR and miR* strands for some miRNA duplexes were similar, indicating similar loading onto miRISC [26]

Non-Canonical miRNA Biogenesis Pathways
IsomiR Biogenesis Pathways
IsomiR Biological Implication
IsomiRs—A Limitless Source of Potentially Novel Biomarkers
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