Figure 3

The complexity of annotating microRNAs from reads mapped to predicted hairpins. (a) Examples of loci that should not be annotated as microRNAs (miRNAs): hairpins with single reads, heterogeneously sized reads, and/or putatively duplexed reads lacking 3' overhangs. (b) Examples of loci with some, but insufficient, evidence for miRNA biogenesis; such loci are worth segregating as candidates pending further study. For instance, depletion of reads from miRNA biogenesis mutants or enrichment in Ago complexes could elevate their status from 'candidate' to 'confident'. It is also worth considering that candidate miRNA hairpins with relatively imprecise processing patterns may represent transitional intermediates in miRNA birth. nt, nucleotide. (c) Confident miRNA hairpins generate relatively precise miRNA/miRNA* duplexes with 3' overhangs. As datasets grow, it is often possible to observe cloned terminal loops or 5'/3' fragments (frag) of the primary miRNA (pri-miRNA) base, whose phasing with miRNA/miRNA* termini provide stringent evidence for in vivo cleavage reactions. Note that the vertebrate locus mir-451 matures by direct hairpin cleavage by Ago2, and not via a miRNA/miRNA* intermediate; thus, the criteria outlined in this figure are not applicable to mir-451-class substrates.