Loop-mediated biogenesis of zebrafish miR-144. (A) Scheme for analysis of wildtype and mutant Dr-pre-mir-144 hairpins after injection into 1-cell zebrafish embryos. Mutations of the entire loop (LM), the 3′ loop sequence (LM3), or of a single bulged nucleotide inferred to mediate apical loop reshaping (LM7) block conversion of pre-mir-144 into miR-144-3p. (B) Activity assay of pre-mir-144 variants. (C) Loop mutant versions of pre-mir-144 are defective in repression of a miR-144 sensor. (D) Quantification of the reporter assays. (E–G) Reinforcement of the stable base-pairing in the preferred pre-mir-144 loop secondary structure impairs maturation of miR-144. (E) Variants of zebrafish pre-mir-144 bearing pairs of LNA substitutions, but otherwise retain wildtype sequence. The ‘LNA stem’ variant affects re-shaping of the hairpin structure, but the ‘LNA loop’ variant is predicted to be neutral. (F) Northern blot showing processing of injected pre-mir-144 molecules, showing selective impairment of mature miR-144 from the LNA stem mutant compared to wildtype and LNA loop variant. (G) Quantification of mature miR-144 levels from independent injection and Northern assays, using one-way ANOVA test.
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