MiRNAs Annotation Criteria

With the increasing rate of sRNA discovery, there is an increased requirement for miRNA annotations. A specific criterion has been managed for the purpose of annotations that include experimental as well as computational data. It has been considered that any sRNA should fulfill two conditions to be recognized as miRNA. These two conditions are expression and biogenesis criteria. Where expression criteria stands for the identification of miRNA by cloning or hybridization; biogenesis criteria includes folding of predicted precursors into a defined stem-loop hairpin structure, phylogenetic miRNA conservation, and increased precursor accumulation on decreased

Dicer activity (Ambros et al. 2003). With the development of more sophisticated techniques for miRNAs prediction, it has been observed that some miRNAs followed the criteria but some not. This has led to the generation of new annotation parameters. These parameters are divided as: primary criteria and ancillary criteria.

Primary criterion defines the fundamental features of miRNAs. It states that biogenesis of around 21 nts miRNA/miRNA* duplex should occur by an excision of a qualifying stem-loop precursor. These criteria have been considered as the most necessary and sufficient parameters for miRNAs annotation. In order to fulfill this parameter, both miRNA as well as miRNA* should satisfy the conditions. In case of miRNA*-defi-cient clones, the potential miRNA candidate should be isolated and sequenced from multiple, independent libraries. During sequencing, often very low abundance of one or two sequencing reads of putative miRNA is obtained. In such cases, even RNA gel blots fail to satisfy the qualifying conditions of primary criteria. It is so because detection of sRNAs via blotting is unable to determine whether it is a miRNA, siRNA, or a decay product of larger precursors. To help out in such cases low depth sequencing and extensive blot analysis using multiple probes is utilized.

In addition to primary criteria, certain other features have also been determined for miRNAs annotation. However, satisfaction of ancillary parameters is not essentially required but fulfilling these features would further enhance the significance of miRNAs annotation. Ancillary criteria include miRNAs conservation, their targets, Dicer-like 1 (DCL1) dependence, RNA Dependent RNA polymerases (RDRs) independence. Conservation of stem-loop secondary structure and mature miRNA sequence among lineages is a sufficient proof for miRNAs annotation. Target prediction is not a necessary miRNAs annotation. There could be cases where conserved miRNAs are target-less or nonconserved miR-NAs have well characterized targets. So, in both cases, functional characterization of miRNAs is not required for its annotation. Plants require DCL1 enzyme for miRNAs generation but not in all cases. The dcll mutant has also been reported to possess miRNAs. So, DCL1 dependence cannot be a strict annotation requirement. Similar is with RDRs. RDRs generate dsRNA precursors. These precursors lead to siRNAs biogenesis and hence, RDR dependence is an essential feature of siRNAs. But miRNAs are not synthesized from RDR-generated dsRNA molecules. As a result, RDR independence should not be a necessary condition for miRNAs annotation (Meyers et al. 2008). This can be summed up as: the primary criterion, but not the ancillary, is a kind of eligibility for sRNAs to be annotated as miRNAs.

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