PMC

Ago2 binds and processes pre-miR-451. (A) Immunoprecipitation of FLAG-mAgo2 in wild-type and mutant embryos injected with pre-miR-451 followed by Northern blot analysis to detect bound miR-451. Input (I), supernatant (S), and immunoprecipitate (IP) are indicated. (B and C) In vitro cleavage assay using hAgo2 or hDicer protein and 5′-radiolabeled pre-miR-430 or pre-miR-451. (C) Ago2 processing reactions were treated with (+) or without (−) RNase I to assay protection of the processed hairpin by Ago2. (D to F) Northern blot analyses to detect mature miR-451 after injection with pre-miR-451 (+) [(D) and (E)] or endogenous miR-451 and miR-430 (F). Injection of wild-type mAgo2 but not a catalytically dead mAgo2^D669A rescues pre-miR-451 processing in vivo (E). The processing of miR-451^mm10-11 is strongly reduced. Endogenous pre-miR-451 at 48 hpf is processed in wild type and MZdicer but not in MZago2 mutants. Diagrams for predicted hairpins, cleavage intermediates, mature miR-451 (red), miR-430 (green), and miRNA* (blue) are shown. P³²* indicates that injected hairpins were radiolabeled ().

A Dicer-independent miRNA. (A) Zebrafish pre-miRNAs and duplexes as indicated. pre-miR-430^ago2-hairpin is a miR-430c hairpin that has been mutated and shortened to form a 42-nt hairpin mimicking pre-miR-451 (ago2-hairpin). (B) GFP-reporter mRNA (green) was co-injected at the one-cell stage with control dsRed mRNA (red). The GFP reporter contains three complementary target sites to miR-430 in its 3′-untranslated region. (C) Northern blot to detect miR-430 in wild-type embryos injected with hairpins as indicated. α-Amanitin was co-injected to inhibit transcription of endogenous pri-miR-430. (D) Northern blot to detect 5′-radiolabeled pre-miR-430^ago2-hairpin after in vitro processing by recombinant hAgo2 and hDicer. (E) In vivo assay to rescue miR-430 function in MZdicer mutants. Bright-field and fluorescent images of the dorsal view of the brain after injection of TxRed dextran in the ventricles (right) in 32-hpf embryos. Brain outline (dashed line), mid-hindbrain boundary (green asterisk), and ventricles (red, white asterisk) are shown. Morphogenesis defects are rescued by injection of a Dicer-independent pre-miR-430^ago2-hairpin or a miR-430-duplex but not a Dicer-dependent pre-miR-430.

MZago2 mutants show reduced erythropoiesis. (A) Expression of hemoglobin (brown) visualized by the oxidation of o-dianisidine (o-das) at 48 hpf in the ducts of Cuvier. Hemoglobinized cells accumulate in wild type but are reduced in MZago2 mutants [group II (mild) and group III (severe) reduction of o-das–positive cells]. (B) Percentage of embryos with hemoglobinized cells in MZago2 mutants (n = 61) compared to wild-type embryos (n = 200), showing strongly reduced (group III; light gray) and partially reduced (group II; gray) numbers of o-das(+) cells(χ² test, P < 0.001). (C) Whole-mount in situ hybridization of ago2 expression at 24 hpf. (D) May-Grünwald/Giemsa stain of erythrocytes from wild-type, MZago2 mutants, and MZago2 injected at one-cell stage with various RNAs as shown (+). Erythrocytes are representative of the mean for each group. (E)Scatterplotof the nuclear cytoplasmic ratio (N:C) for each genotype in (D) as a readout of erythrocyte maturation (). Distributions of the N:C ratios in wild-type compared to MZago2 differed significantly (Wilcoxon rank-sum test after Bonferroni correction, P < 10⁻¹⁵). Erythrocyte maturation is rescued by miR-451-duplex (MZago2 and MZago2+ miR-451, P < 10⁻¹⁵) and wild-type mAgo2 (MZago2 and MZago2+mAgo2, P < 10⁻¹⁵) but not catalytically dead mAgo2^D669A (MZago2 and MZago2+mAgo2^D669A, P > 0.1).

MicroRNA analysis in wild type (wt) and in MZdicer and MZago2 mutants. (A and B) Normalized reads from wild type versus MZdicer (A) or MZago2 (B) libraries for all annotated zebrafish miRNAs. Some miRNAs are shown as a reference for enhanced and reduced miRNAs (solid circles); miR-144-5′ (green) and miR-451-5′ (red) are expressed in the same pri-miRNA. (C) Scheme of miR-144/miR-451 genomic loci and predicted secondary structure of both human and zebrafish pre-miR-451 (mature miRNA in red). (D) Total number of reads that matchmiR-451inwildtype, MZdicer, and MZago2. Nontemplated uridines are shown in red. (E)Domain organization of Ago2. The 90-nt deletion (Δ90) results in a predicted truncated protein lacking two of three catalytic residues. Amino acid positions are based on the mammalian Ago2. (F) Northern blot of embryos to detect slicer cleavage of an injected GFP target mRNA with three complementary targets to miR-1 (3×PT-miR-1) in the presence (+) or absence (−) of miR-1 (). Slicer activity is indicated by the higher-mobility product () ().