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RESEARCH COMMUNICATION
1 The Laboratory for Lymphocyte Signaling, The Rockefeller University, New York, New York 10021, USA; 2 Wellcome Trust/Cancer Research, UK Gurdon Institute and Department of Biochemistry, University of Cambridge, Cambridge CB2 1QN, United Kingdom; 3 Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, United Kingdom
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[Keywords: Ago2; Slicer; microRNA; hematopoeisis]
Received April 30, 2007; revised version accepted June 5, 2007.
; Hutvagner et al. 2001; Ketting et al. 2001; Knight and Bass 2001), whereas Ago proteins (Ago1–4) bind miRNA (Lingel et al. 2003; Song et al. 2003; Yan et al. 2003) and mediate gene silencing (Hammond et al. 2001; Hutvagner and Zamore 2002). The mechanism of RISC-mediated gene silencing depends on the degree of complementarity between the miRNA and its target. Binding of miRNA–RISC to a partially complementary mRNA results in silencing through the inhibition of translation or mRNA degradation by catabolism within processing bodies (for review, see Pillai et al. 2007). In turn, direct cleavage or "slicing" of mRNA that is solely catalyzed by Ago2 (Liu et al. 2004; Meister et al. 2004; Song et al. 2004) requires perfect or near-perfect complementarity between the miRNA and the target (Hutvagner and Zamore 2002; Doench et al. 2003). To date, a single mammalian miRNA has been shown to direct Ago2-mediated slicing (Mansfield et al. 2004; Yekta et al. 2004); however, the ability of Ago2 to slice significantly mismatched targets in vitro indicates that the complementarity rules may not be as stringent as suggested (Martinez and Tuschl 2004). While the function of Ago2’s Slicer activity as the catalytic engine that powers RNA interference (RNAi) is understood, the general in vivo physiological importance of Ago2 and its endonuclease activity within the miRNA pathway remains largely unknown. |
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; Cobb et al. 2005; Fazi et al. 2005; Muljo et al. 2005) makes this process attractive for the investigation of the physiological significance of Ago2. We therefore studied the development of the lymphoid and erythroid lineages derived from Ago2–/– bone marrow cells. The latter cells were produced by inducible inactivation of the Ago2 gene in bone marrow progenitors followed by the generation of Ago2–/– hematopoietic system in lethally irradiated wild-type mice (Supplementary Fig. S1). This approach enables the analysis of the bone marrow cell-intrinsic Ago2 function. Mice reconstituted with Ago2–/– or control Ago2fl/fl bone marrow cells are hereafter referred to as Ago2–/– or Ago2fl/fl mice, respectively. Analysis of mice 6–8 wk after reconstitution revealed that Ago2–/– bone marrow cells give rise to hematopoietic cells of various lineages (data not shown). However, the differentiation of the B lymphoid as well as erythroid lineages diverges significantly from the wild-type developmental pattern. Deficiency in Ago2 does not interfere with the generation of early pro-B (B220loCD43+IgM–) cells in the bone marrow, but affects further pre-B (B220loCD43–IgM–) cell differentiation and the subsequent generation of peripheral B cells (Fig. 1A; Table 1). Among various hematopoietic lineages in the bone marrow, erythroid cells appear to be most affected. The maturation of the Ago2–/– erythroid precursors is severely impaired. This developmental defect is characterized by a dramatic increase in the frequencies and numbers of immature Ter119hi, CD71hi basophilic erythroblasts and decrease in frequencies and numbers of Ter119hi, CD71neg mature orthochromatophilic erythroblasts in the bone marrow and spleens of Ago2–/– mice (Fig. 1B; Table 1). Ago2–/– mice display erythroid hyperplasia in both the bone marrow and spleen (Fig. 1B). The combination of ineffective erythropoiesis and erythroid hyperplasia results in splenomegaly in Ago2–/– mice (Fig. 1D). The defective erythropoiesis in the absence of Ago2 fails to generate fully functional red blood cells (RBCs). Ago2–/– mice develop severe anemia characterized by a reduction in the overall number of erythrocytes as well as the amount of hemoglobin per erythrocyte (Fig. 1C). Ago2–/– RBCs, but not control erythrocytes, contained Heinz bodies, inclusion bodies that contain Hb precipitates (Fig. 1E). The same phenotype was observed at 3 mo after reconstitution (data not shown). Therefore, these alterations do not represent a stress response to the transfer but rather reflects the long lasting consequence of Ago2 deficiency.
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) and in vivo (Supplementary Fig. S2). Ago2–/– bone marrow cells were complemented with exogenous wild-type or Slicer-inactive Ago2 and used to generate bone marrow chimeras. Exogenous wild-type Ago2 and Slicer-inactive Ago2 were expressed at similar levels in both the bone marrow and bone marrow-derived splenic cells of reconstituted mice (Supplementary Fig. S3A). Most importantly, expression of either wild-type Ago2 or Slicer-inactive Ago2 could cure the impaired erythropoiesis and anemia caused by Ago2 deficiency. The expression of Slicer-inactive Ago2 restored the wild-type pattern of erythroid development in the bone marrow and spleen, rescuing ineffective erythropoiesis, erythroid hyperplasia, and splenomegaly (Fig. 2A; Supplementary Fig. S3C). In addition, expression of the Slicer-inactive Ago2 in Ago2–/– cells restored erythroid parameters and prevented Heinz body formation to the same degree as exogenous wild-type Ago2 (Fig. 2C; Supplementary Fig. S3B). Similar to the erythroid cells, expression of Slicer inactive Ago2 was able to support normal B-cell development in the bone marrow (Fig. 2B). These results show the dispensable role of Ago2’s slicer function in regulation of erythropoiesis and B-cell lymphopoiesis.
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; Gregory et al. 2005; Haase et al. 2005; Maniataki and Mourelatos 2005; Meister et al. 2005; Lee et al. 2006). To address the role of Ago2 in regulation of miRNA homeostasis, we measured the expression levels of miRNAs in Ago2–/– erythroblasts. Using miRNA microarray analysis, we observed a reduction in the levels of miRNAs in Ago2–/– erythroblasts compared with Ago2fl/fl controls (Fig. 3A). This global reduction in miRNA expression was confirmed using quantitative RT–PCR (qRT–PCR) on two representative miRNAs, and is exemplified by a five- to eightfold reduction of the respective miRNAs in Ago2–/– erythroblasts (Fig. 3A). The reduced miRNA in the absence of Ago2–/– is a general feature of Ago2 deficiency and not a result of defective erythroid differentiation per se. Similar to Ago2–/– erythroblasts both Ago2–/– fibroblasts and hepatocytes display a reduction in miRNA expression levels (Fig. 3B,C).
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). Therefore, the accumulation of pre-miR-24 and pre-miR-199a in Ago2–/– fibroblasts, two pre-miRNAs that are readily detectible in wild-type fibroblasts, indicates a crucial role for Ago2 in miRNA biogenesis (Fig. 4A). The impaired miRNA biogenesis is independent of Ago2’s Slicer activity, as complementation of Ago2–/– cells with slicer-inacitve Ago2 restores miRNA biogenesis (Fig. 4B,C). The dispensability of Ago2’s Slicer activity in miRNA biogenesis is supported by the fact that Slicer-inactive Ago2 interacts with Dicer in vivo, and this protein complex possesses pre-miRNA processing activity in vitro (Maniataki and Mourelatos 2005). The defective miRNA biogenesis cannot be attributed to the reduced expression of Dicer that is expressed at normal levels in Ago2–/– cells (Fig. 4D). These data suggest that absence of Ago2 from RISC hampers either access of Dicer to pre-miRNA or reduces its overall enzymatic activity. The interaction of Ago2 with Dicer through the TRBP and PACT couples miRNA biogenesis with the loading of RISC. It is possible that the absence of Ago2 impairs RISC function in a fashion similar to RISC malfunction caused by reduced expression of PACT or TRBP (Chendrimada et al. 2005; Gregory et al. 2005; Haase et al. 2005; Lee et al. 2006). We demonstrate that Ago2 has an earlier function within RISC in facilitating miRNA biogenesis, this is also supported by the fact that both TRBP and PACT deficient mice, in contrast to Ago2, are viable and display minor phenotypic alterations (Zhong et al. 1999; Rowe et al. 2006). The reduced levels of miRNAs in Ago2–/– cells indicate that the other Argonautes cannot compensate for the loss of Ago2. Indeed, the negligible role of these proteins in miRNA homeostasis and hematopoiesis was confirmed in Ago1–/– or Ago3–/– cells and mice, respectively (Fig. 4E,F; Supplementary Fig. S4; U. Koenig, D. O’Carroll, and A. Tarakhovsky, in prep.)
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The Ago2 locus contains 16 exons. The targeting strategy allows Cre-mediated deletion of exons 9–11. This deletion excludes in-frame splicing of exon 8 to exons 12–16, and leads to the functional inactivation of Ago2 due to partial deletion of the PAZ domain and loss of RNase activity. In addition, according to the rules of RNA surveillance (Hentze and Kulozik 1999), the in-frame stop codons generated by frame-shift mutations introduced by the deletion of exons 9–11 is likely to destabilize the resulting primary transcript RNA and prevent production of a truncated Ago2 protein. The Ago2 gene targeting vector (pDTA–TK–Ago2) carries a neomycin resistance (neor) gene flanked with two frt sites, a loxP site (neor–frt2–loxP cassette) located 3' of exon 11 and a loxP site (5'loxP) within the intron between exons 8 and 9. The Ago2 targeting construct was transfected into E14.1 embryonic stem (ES) cells. Southern blotting of the individual ES cell clone-derived genomic BamHI-digested DNA with probe 12i was used to identify homologous recombinants. A 4.2-kb DNA fragment corresponds to the wild-type Ago2 locus, and integration of the neor–frt2–loxP cassette 3' of exon 11 introduces an additional BamHI site, thus increasing the size of the BamHI DNA fragment recognized by probe 12i to 5.0 kb. Targeted ES cells were used to generate mice heterozygous for the Ago2-targeted allele. These mice were then crossed to the FLP-expressing transgenic mice (FLPeR) (Farley et al. 2000) to remove the frt-flanked neor cassette, resulting in the generation of Ago2 loxP allele (Ago2 fl allele).
Generation of Ago2–/– bone marrow and analysis of bone marrow chimeras
To inactivate the Ago2 gene in the bone marrow, Ago2fl/fl mice (6–12 wk of age) that carry the MxCre transgene (Kuhn et al. 1995) (Ago2fl/fl;MxCre mice) received three i.p. injections of 250 µg per mouse of pI:pC, spaced 3 d apart. Mice were sacrificed no earlier than 10 d after the last injection and bone marrow cells were isolated and transferred into lethally irradiated (875 R) C57/Bl6 mice (6 x 106 to 7 x 106 cells per recipient). Bone marrow cells derived from Ago2fl/fl mice treated with pI:pC as described above were used as control. Bone marrow-reconstituted mice were maintained on medicated water (1000 U/mL polymixin B, 1.1 g/L neomycin sulfate) for 4 wk after reconstitution and were analyzed 6–8 wk or 3 mo after transfer. Isolation of spleen and bone marrow and subsequent FACS analysis were performed as described (Socolovsky et al. 2001; Mecklenbrauker et al. 2002). Blood parameters were analyzed using flow cytometry-based hematology (Bayer, Advia 120).
Generation of an Ago2 antibody
A synthetic peptide encompassing the first 25 amino acids of mouse Ago2 was coupled to KLH and was used as an immunogen in rabbits. Crude serum was obtained after the first boost and affinity-purified. This serum was then used in a 1:1000 dilution for Western blot analysis.
Cell extract preparation and immunodetection of Ago2
Cells were lysed in 150 mM NaCl, 20 mM Tris (pH 7.5), 1 mM EDTA, 1% Triton X-100, and Complete protease inhibitors (Roche); after 10-min centrifugation at 13,000g, the supernatant was collected and used as extract for immunoblotting, using standard protocols.
Retroviral transduction of Ago2–/– cells
The coding sequences of Ago2 and Ago2D669A were inserted into the HpaI site of MigR retroviral vector, and recombinant retroviruses were produced and used to infect Ago2–/– bone marrow or fibroblasts as described (Pear et al. 1998). Reconstitution of lethally irradiated mice was performed as above.
Derivation and manipulation of mouse embryonic fibroblast (MEF) cell lines
Two Ago2fl/fl MEF cell lines (MEF lines 4 and 9) were derived from E12.5 embryos, transformed with SV40 large T-antigen-expressing retrovirus, and propagated under standard culture conditions. To generate Ago2–/– MEFs, both Ago2fl/fl MEF clones were infected with Cre-expressing adenovirus (AdenoCre; Vector Biolabs). For the miRNA-guided cleavage assay, the same assay as described in Pillai et al. (2005) was employed.
MiRNA expression analysis
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer’s instructions. MiRNA array profiling was performed as described (Miska et al. 2004). For clustering, only miRNAs with expression levels at least five times above background were selected. Expression data were log-transformed, gene-centered (mean), and normalized, and hierarchical clustering was performed using a correlation matrix and centroid linkage using the Cluster 3.0 algorithm. qRT–PCR expression analysis of miRNAs was performed using mirVana qRT–PCR miRNA detection kit (Ambion) and Roche LightCycler 480. Northern blotting of miRNAs was performed as described (Lau et al. 2001).
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E-MAIL donal.ocarroll{at}embl-monterotondo.it; FAX 39-060900-91406.
6 E-MAIL tarakho{at}mail.rockefeller.edu; FAX (212) 327-8258.
Supplemental material is available at http://www.genesdev.org.
Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.1565607
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