Abstract
As more small RNA sequencing libraries are becoming available, it clearly emerges that microRNAs (miRNAs) are highly heterogeneous both in length and sequence. In comparison to canonical miRNAs, miRNA isoforms (termed as “isomiRs”) might exhibit different biological properties, such as a different target repertoire, or enhanced/reduced stability. Nonetheless, this layer of information has remained largely unexplored due to the scarcity of small RNA NGS-datasets and the absence of proper analytical tools. Here, we present a workflow for the characterization and analysis of miRNAs and their variants in next-generation sequencing datasets. IsomiRs can originate from an alternative dicing event (“templated” forms) or from the addition of nucleotides through an enzymatic activity or target-dependent mechanisms (“non-templated” forms). Our pipeline allows distinguishing canonical miRNAs from templated and non-templated isomiRs by alignment to a custom database, which comprises all possible 3′-, 5′-, and trimmed variants. Functionally equivalent isomiRs can be grouped together according to the type of modification (e.g., uridylation, adenylation, trimming …) to assess which miRNAs are more intensively modified in a given biological context. When applied to the analysis of primary epithelial breast cancer cells, our methodology provided a 40% increase in the number of detected miRNA species and allowed to easily identify and classify more than 1000 variants. Most modifications were compatible with templated IsomiRs, as a consequence of imprecise Drosha or Dicer cleavage. However, some non-templated variants were consistently found either in the normal or in the cancer cells, with the 3′-end adenylation and uridylation as the most frequent events, suggesting that miRNA post-transcriptional modification frequently occurs. In conclusion, our analytical tool permits the deconvolution of miRNA heterogeneity and could be used to explore the functional role of miRNA isoforms.
Highlights
MicroRNAs, a small (18–25 nt long), evolutionarily conserved class of non-coding RNAs, are important regulators of transcriptional programs by silencing the expression of a multitude of target mRNAs at a post-transcriptional level (Bartel, 2009)
One strand is retained in the RNA-induced silencing complex (RISC), usually the one with unstable base-pairing at its 5 -end, and it mediates target repression through base complementarity between the miRNA “seed region” and the miRNA responsive elements (MRE), mostly located at the 3 untranslated region (3 UTR) of target genes (Bartel, 2009)
IsomiRs were considered as sequencing artifacts, but a growing body of evidence revealed that isomiRs are actual miRNA variants that can exert a biological activity
Summary
MicroRNAs (miRNAs), a small (18–25 nt long), evolutionarily conserved class of non-coding RNAs, are important regulators of transcriptional programs by silencing the expression of a multitude of target mRNAs at a post-transcriptional level (Bartel, 2009). The biogenesis of miRNAs typically requires a nuclear cleavage of the primary transcript by the Drosha/DGCR8 complex and a cytoplasmic cleavage of the hairpin-folded precursor miRNA (premiRNA) by Dicer [reviewed in Krol et al (2010)]. The product of this cleavage is usually a mature 21/22 bp miRNA duplex, which is loaded onto the RNA-induced silencing complex (RISC) to function in the miRNA silencing mechanism (Gregory et al, 2005). IsomiRs could be generated by post-transcriptional modifications due to enzymatic activity, which could either add or remove specific nucleotides to miRNA ends These miRNA variants are known as “non-templated isomiRs,” with sequence imperfectly matching their pre-miRNA. This is likely due to fact that a 5 -end www.frontiersin.org
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
