A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity - PubMed (original) (raw)

. 2010 Jun 25;328(5986):1694-8.

doi: 10.1126/science.1190809. Epub 2010 May 6.

Huiling Xue, David W Taylor, Heather Patnode, Yuichiro Mishima, Sihem Cheloufi, Enbo Ma, Shrikant Mane, Gregory J Hannon, Nathan D Lawson, Scot A Wolfe, Antonio J Giraldez

Affiliations

A novel miRNA processing pathway independent of Dicer requires Argonaute2 catalytic activity

Daniel Cifuentes et al. Science. 2010.

Abstract

Dicer is a central enzyme in microRNA (miRNA) processing. We identified a Dicer-independent miRNA biogenesis pathway that uses Argonaute2 (Ago2) slicer catalytic activity. In contrast to other miRNAs, miR-451 levels were refractory to dicer loss of function but were reduced in MZago2 (maternal-zygotic) mutants. We found that pre-miR-451 processing requires Ago2 catalytic activity in vivo. MZago2 mutants showed delayed erythropoiesis that could be rescued by wild-type Ago2 or miR-451-duplex but not by catalytically dead Ago2. Changing the secondary structure of Dicer-dependent miRNAs to mimic that of pre-miR-451 restored miRNA function and rescued developmental defects in MZdicer mutants, indicating that the pre-miRNA secondary structure determines the processing pathway in vivo. We propose that Ago2-mediated cleavage of pre-miRNAs, followed by uridylation and trimming, generates functional miRNAs independently of Dicer.

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Figures

Fig. 1

Fig. 1

MicroRNA analysis in wild type (wt) and in MZ_dicer_ and MZ_ago2_ mutants. (A and B) Normalized reads from wild type versus MZ_dicer_ (A) or MZ_ago2_ (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, MZ_dicer_, and MZ_ago2_. 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 (7). Slicer activity is indicated by the higher-mobility product (fig. S1) (18).

Fig. 2

Fig. 2

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 mAgo2D669A rescues pre-miR-451 processing in vivo (E). The processing of miR-451mm10-11 is strongly reduced. Endogenous pre-miR-451 at 48 hpf is processed in wild type and MZ_dicer_ but not in MZ_ago2_ 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 (18).

Fig. 3

Fig. 3

MZ_ago2_ 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 MZ_ago2 mutants [group II (mild) and group III (severe) reduction of o-das–positive cells]. (B) Percentage of embryos with hemoglobinized cells in MZ_ago2_ 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, MZ_ago2_ mutants, and MZ_ago2_ 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 (17). Distributions of the N:C ratios in wild-type compared to MZ_ago2_ differed significantly (Wilcoxon rank-sum test after Bonferroni correction, _P_ < 10−15). Erythrocyte maturation is rescued by miR-451-duplex (MZ_ago2_ and MZ_ago2_+ miR-451, _P_ < 10−15) and wild-type mAgo2 (MZ_ago2_ and MZ_ago2_+mAgo2, _P_ < 10−15) but not catalytically dead mAgo2D669A (MZ_ago2_ and MZ_ago2_+mAgo2D669A, _P_ > 0.1).

Fig. 4

Fig. 4

A Dicer-independent miRNA. (A) Zebrafish pre-miRNAs and duplexes as indicated. pre-miR-430ago2-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-430ago2-hairpin after in vitro processing by recombinant hAgo2 and hDicer. (E) In vivo assay to rescue miR-430 function in MZ_dicer_ 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-430ago2-hairpin or a miR-430-duplex but not a Dicer-dependent pre-miR-430.

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