A dicer-independent miRNA biogenesis pathway that requires Ago catalysis

Abstract

The nucleolytic activity of animal Argonaute proteins is deeply conserved, despite its having no obvious role in microRNA-directed gene regulation. In mice, Ago2 (also known as Eif2c2) is uniquely required for viability, and only this family member retains catalytic competence. To investigate the evolutionary pressure to conserve Argonaute enzymatic activity, we engineered a mouse with catalytically inactive Ago2 alleles. Homozygous mutants died shortly after birth with an obvious anaemia. Examination of microRNAs and their potential targets revealed a loss of miR-451, a small RNA important for erythropoiesis. Though this microRNA is processed by Drosha (also known as Rnasen), its maturation does not require Dicer. Instead, the pre-miRNA becomes loaded into Ago and is cleaved by the Ago catalytic centre to generate an intermediate 3' end, which is then further trimmed. Our findings link the conservation of Argonaute catalysis to a conserved mechanism of microRNA biogenesis that is important for vertebrate development.

Figure 1. Ago2 is essential for extra-embryonic development

a, LacZ whole-mount staining of embryonic day 9.5 embryos bearing the Ago2^gt or Ago1^gt gene trap beta-galactosidase reporter alleles and wild-type controls (WT ctrl). b, Allelic combination in Ago2 insertional mutant embryos, with the structure of each allele shown. HPRT, hypoxanthine-guanine phosphoribosyl transferase gene; NEO, neomycin resistance gene; PURO, puromycin resistance gene. c, Example wild-type (WT) and mutant (Ago2m^mc/gt) embryos from heterozygous intercrosses. Left panel: embryonic day 10.5 embryos within their embryonic yolk sac and placentas. Right panel: embryos dissected from their extra-embryonic components. d, Representative embryonic day 12.5 chimaeric embryo developed from Ago2 null ES cells aggregated with wild-type tetraploid embryos. From left to right: whole chimaeric embryonic conceptus (Ago2^mc/gt:WT), beta-galactosidase staining of the whole embryo showing contribution of Ago2 null ES cells (Ago2^mc/gt), beta-galactosidase staining of the placenta showing contribution of the ES cells to the vasculature invading the placental labyrinth (Ago2^mc/gt:WT).

Figure 2. Ago2 catalysis is essential for development

a, Representative neonates from Ago2^ADH heterozygous inter-crosses. WT, wild type; Mut, Ago2 homozygous mutant. b, Peripheral blood count of litter mates from WT and Mut. Data are the mean ± s.d. *t-test (unequal variance for embryonic day 18.5 time point P = 0.035, equal variance for the birth time point P = 1.95 × 10²⁷). c, Representative FACS analysis using CD71/Ter-119 erythroid population marking of individual bone marrow samples of mutant versus wild-type litter mates. Three independent pairs from three different litters showed virtually identical profiles. APC, allophycocyanin; FITC, fluorescein isothiocyanate; FACS, fluorescence-activated cell sorting.

Figure 3. Mature miR-451 expression depends on Ago2 catalysis

a, Scatter plot of miRNA reads in wild-type versus mutant fetal liver. b, Quantitative RT–PCR of primary transcript levels of miR-144 and miR-451 in wild-type and mutant liver samples. Data are the mean of three biological replicates ± s.d. *t-test with equal variance P > 0.05. c, The unique structure of the miR-451 hairpin compared to miR-144 with the miRBase annotation of mature miR-451 and miR-144 mapped to the predicted secondary hairpin structure shown. Guide strand in red and passenger strand in blue.

Figure 4. Non-canonical biogenesis of miR-451

a, Effect on mature miRNA levels of Drosha conditional ablation in Drosha flox/flox Cre-ER MEFs. b, In vitro processing of miR-451 and miR-144 primary transcripts by Drosha immunoprecipitates (IP). Pre-miR-144 and pre-miR-451 are indicated with their corresponding expected sizes. nt, nucleotides. Additional fragments released by possible Drosha processing of the 5′ miR-451 flank are indicated with asterisks. Flanks are indicated with arrowheads. c, Northern blots for confirmation of in vitro Drosha processing assays. d, Effect on mature miRNA levels of Dicer conditional ablation in Dicer flox/flox Cre-ER ES cells. e, Effect on mature miRNA levels in Dicer null stable ES cells. f, Northern blot of mature miR-451 expression in Dicer null stable ES cells. U6 is used as a loading control. MSCV, murine stem cell virus.

Figure 5. Ago2 catalysis is required for miR-451 biogenesis

a, Left panels, northern blot on total RNA from WT and mutant livers probing for the miR-451 guide strand and the passenger arm of the hairpin (indicated). Let-7c is used as a loading control. Right panels, northern blots of Ago2 and IgG control immunoprecipitates from WT and mutant liver extracts with the indicated probes. b, miRNA read length distribution for the indicated miRNA from deep sequencing of WT and mutant livers. c, prediction of a miR-451 Ago2 cleavage site. Top, miR-451 3′ end heterogeneity. Bottom, predicted cleavage site at the thirtieth phosphodiester bond of pre-miR-451. d, Left gel, in vitro cleavage assay of pre-miR-451 by an Ago2 immunoprecipitate. Right gel, confirmation of the 3′ end character of the Ago2 cleavage product using beta elimination and 3′ end ligation reactions. β, beta-eliminated; UT, untreated. e, Immunoprecipitation-northern blot indicating presence of the mature form of miR-451 in Ago2 complexes and loading of pre-miR-451 without processing in Ago1 complexes. f, Left, schematic depictions of the pre-let-7c-miR-451 mimic hairpin compared to the native pre-let-7c. Guide strand in red and passenger strand in blue. Right, FACS analysis for GFP in the indicated samples. Cells were co-transfected with phycoerythrin (PE)–siRNA control. PE-positive cells (10⁵) were gated and analysed for GFP expression. siglo is a fluorescently labelled siRNA used as a transfection control and dox is doxycycline.