Figure 4

Identification of ac-pre-miRNA candidates. (a) Sequence logo profile of hsa-miR-451 showing 5' arm dominance, almost no variation in the 3' end of the mature miRNA, and a trail of 3' isomiR extensions. (b) Schematic diagram illustrating the generation of end-site (5p-ac-pre-miRNA) and start-site (3p-ac-pre-miRNA) isomiRs via AGO2-mediated cleavage of a pre-miRNA. Dashed lines indicate variable regions. (c) Sequence logo profile for hsa-miR-31, which was identified by our analysis as a candidate for biogenesis through the ac-pre-miRNA pathway. Three representative tissues are shown. (d) Wild-type (WT) zebrafish whole embryo versus mutant AGO2 inactive (MZAgo2) whole embryo miRNA and isomiR expression. Each point represents a separate isomiR or miRNA. The thick blue line is the perfect correlation line, the thin blue lines are ± 2-fold visual guides. Red dots indicate isomiRs/miRNAs derived from candidate ac-pre-miRNA hairpins. A bias towards down-regulation in AGO2 mutants is evident in this plot. (e) Box and whiskers plots showing the expression of all isomiRs/miRNAs deriving from ac-pre-miRNA candidates in wild-type (WT) and mutant (MZago2) miRNA-seq samples; 75% of ac-pre-miRNA candidate molecules are not expressed in the mutant sample.