Trimming Is Dispensable for Target Silencing In Vivo

(A) The sequences and structures of pre-miR-430, miR-430 duplex, and pre-miR-430^{Ago2 hairpin} used in zebrafish experiments.

(B) Inhibition of trimming by three phosphorothioate linkages in zebrafish embryos. Pre-miR-430^{Ago2 hairpin} with or without 3× PS was injected, and RNAs were analyzed by northern blotting.

(C) Schematic representation of the reporter mRNAs with 3× target sites used in (D) and (E).

(D and E) Reporter assays for the 3× imperfect target sites by pre-miR-430^{Ago2 hairpin} with or without 3× PS. (D) GFP reporter with DsRed control. (E) Firefly luciferase reporter with Renilla luciferase control. The Fluc/Rluc luminescence was normalized to the value of mock injection. The mean values ± SDs from three independent experiments are shown. Trimming was dispensable for reporter silencing in zebrafish embryos.

(F) Trimming is not required to rescue the brain morphologic defects in MZdicer zebrafish embryos. MZdicer embryos were injected miR-430 duplex, pre-miR-430^{Ago2 hairpin}, or pre-miR-430^{Ago2 hairpin} 3× PS and analyzed at 30 hpf. See also Figure S4.

Trimming is not essential to rescue the MZdicer phenotype, Related to Figure 5.

Injection of miR-430 duplex, pre-miR-430Ago2 hairpin or pre-miR-430Ago2 hairpin 3Å~PS rescued the morphological defects of MZdicer mutant embryos. Three representative embryos for each condition are shown.