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The miRNA machinery targets Mei-P26 and regulates Myc protein levels in the Drosophila wing

2010; Springer Nature; Volume: 29; Issue: 10 Linguagem: Inglês

10.1038/emboj.2010.69

1460-2075

Héctor Herranz, Xin Hong, Lídia Pérez, Ana Ferreira, Daniel Olivieri, Stephen M. Cohen, Marco Milán,

Circular RNAs in diseases

Article16 April 2010free access The miRNA machinery targets Mei-P26 and regulates Myc protein levels in the Drosophila wing Héctor Herranz Héctor Herranz Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Xin Hong Xin Hong Temasek Life Sciences Laboratory, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore Search for more papers by this author Lidia Pérez Lidia Pérez Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Ana Ferreira Ana Ferreira Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Daniel Olivieri Daniel Olivieri Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Stephen M Cohen Stephen M Cohen Temasek Life Sciences Laboratory, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore Search for more papers by this author Marco Milán Corresponding Author Marco Milán Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Héctor Herranz Héctor Herranz Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Xin Hong Xin Hong Temasek Life Sciences Laboratory, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore Search for more papers by this author Lidia Pérez Lidia Pérez Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Ana Ferreira Ana Ferreira Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Daniel Olivieri Daniel Olivieri Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain Search for more papers by this author Stephen M Cohen Stephen M Cohen Temasek Life Sciences Laboratory, National University of Singapore, Singapore Department of Biological Sciences, National University of Singapore, Singapore Search for more papers by this author Marco Milán Corresponding Author Marco Milán Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain ICREA, Barcelona, Spain Search for more papers by this author Author Information Héctor Herranz1, Xin Hong2,3, Lidia Pérez1, Ana Ferreira1, Daniel Olivieri1, Stephen M Cohen2,3 and Marco Milán 1,4 1Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Barcelona, Spain 2Temasek Life Sciences Laboratory, National University of Singapore, Singapore 3Department of Biological Sciences, National University of Singapore, Singapore 4ICREA, Barcelona, Spain *Corresponding author. Cell and Developmental Biology Programme, Institute for Research in Biomedicine, Baldiri i Reixac, 10, Barcelona 08028, Spain. Tel.: +349 3403 4902; Fax: +349 3403 7109; E-mail: [email protected] The EMBO Journal (2010)29:1688-1698https://doi.org/10.1038/emboj.2010.69 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info MicroRNAs (miRNAs) have been implicated in cell-cycle regulation and in some cases shown to have a role in tissue growth control. Depletion of miRNAs was found to have an effect on tissue growth rates in the wing primordium of Drosophila, a highly proliferative epithelium. Dicer-1 (Dcr-1) is a double-stranded RNAseIII essential for miRNA biogenesis. Adult cells lacking dcr-1, or with reduced dcr-1 activity, were smaller than normal cells and gave rise to smaller wings. dcr-1 mutant cells showed evidence of being susceptible to competition by faster growing cells in vivo and the miRNA machinery was shown to promote G1–S transition. We present evidence that Dcr-1 acts by regulating the TRIM-NHL protein Mei-P26, which in turn regulates dMyc protein levels. Mei-P26 is a direct target of miRNAs, including the growth-promoting bantam miRNA. Thus, regulation of tissue growth by the miRNA pathway involves a double repression mechanism to control dMyc protein levels in a highly proliferative and growing epithelium. Introduction Regulation of gene expression at the transcriptional level has a central role in development and physiology; however, the relevance of post-transcriptional gene regulation is increasingly recognized. MicroRNAs (miRNAs), endogenous small non-coding RNAs, 22 nucleotides long, that repress target transcripts (Flynt and Lai, 2008), confer a novel layer of post-transcriptional regulation. Dicer-1 (Dcr-1) is a crucial element for miRNA biogenesis (Lee et al, 2003). Therefore, impairing Dcr-1 activity provides a means to assess the role of the miRNA pathway in a given biological process. Loss of dcr-1 produces defects in Drosophila and vertebrate stem cell maintenance and causes a delay in G1–S transition in these cells (Hatfield et al, 2005; Jin and Xie, 2007; Wang et al, 2007). In the developing mouse limb, loss of dcr-1 leads to growth defects (Harfe et al, 2005). The main effectors mediating the activity of Dcr-1 in these processes have not been identified. The wing imaginal disc of Drosophila is a very suitable model system to analyse at a cellular level the role of miRNAs in a highly proliferative epithelium and to identify such effectors. The fly wing primordium arises as a group of 30–40 cells in the embryonic ectoderm that proliferates during 5 days to reach a final size of around 50 000 cells and gives rise after metamorphosis to the adult wing (García-Bellido and Merriam, 1971; Madhavan and Schneiderman, 1977). Here we have analysed the role of Dcr-1 in growth control in the developing wing. Dcr-1 is required for cell and tissue growth, promotes G1–S transition and dcr-1 mutant cells are eliminated by a process of cell competition. We present evidence that the dMyc proto-oncogene (Johnston et al, 1999) contributes to the role of the miRNA pathway in these processes. TRIM32, the mouse orthologue of Drosophila Mei-P26 (Page et al, 2000), has been shown to show ubiquitin ligase activity, bind to c-Myc and target it for degradation (Schwamborn et al, 2009). We present evidence that the miRNA machinery, acting through Mei-P26, regulates dMyc activity, and as a consequence regulates cell and tissue growth rates and E2F activity. Mei-P26 is a direct target of the growth-promoting bantam miRNA. This study identifies the elements of an integrated regulatory network involving miRNAs, regulators of cell growth and of cell proliferation. Results and discussion Reduced growth in the absence of Dcr-1 We first used the Gal4/UAS technique to express an RNAi construct of dcr-1 (dcr-1RNAi) to reduce Dcr-1 activity in specific territories within the developing wing. To assess the efficacy of the dcr-1RNAi transgene, we measured the levels of 11 miRNAs known to be expressed in wing imaginal discs by quantitative PCR. Nine of these miRNAs were reduced (Figure 1A). We also analysed the capacity of dcr-1RNAi to reduce the processing of the bantam miRNA in the wing cells. Expression of dcr-1RNAi reduced bantam activity as visualized by increased levels of a bantam activity sensor (Brennecke et al, 2003) in the wing imaginal disc (Supplementary Figure S1). The magnitude of this effect was less than that observed in clones of cells mutant for a null allele of dcr-1 (dcr-1Q1147X (Lee et al, 2004), Supplementary Figure S1). Expression of a sensor lacking the bantam target sites (Brennecke et al, 2003) was not affected upon dcr-1RNAi expression (Supplementary Figure S1). Thus, dcr-1RNAi expression leads to an intermediate condition where Dcr-1 activity and miRNA levels are reduced but not eliminated. Expression of dcr-1RNAi caused an autonomous reduction in cell size in the adult wing (Figure 1B and G; see also Supplementary Table S1). As a consequence, the size of the tissue that expressed dcr-1RNAi was also reduced (Figure 1C–F). These effects were rescued by co-expression of a wild-type form of dcr-1 (Figure 1D, F and G). Decreasing the amount of dcr-1 by 50% enhanced the size defects caused by dcr-1RNAi expression (Figure 1D) and the viability of these flies was drastically reduced (data not shown). Similar adult cell size defects were observed in clones of cells mutant for a null allele of dcr-1 (dcr-1Q1147X, Figure 1H and I). Figure 1.Reduced cell growth in the absence of Dcr-1 activity. (A) Quantitative PCR experiment comparing the relative level of mature miRNAs between the anterior (a, white bars) and posterior (p, blue bars) compartments of en-gal4; UAS-dicer-1RNAiUAS-GFP late third instar wing discs. The reference gene U27 was used for data normalization for RNAs from different compartments. The relative levels of miRNAs in the a compartment were set to ‘1’ (white bars). en-gal4 drives expression of dicerRNAi and GFP in the p compartment. The y-axis shows the percentages of miRNA expression level relative to the a compartment. A wing disc is depicted to visualize a (white) and p (blue) compartments of a mature wing disc. (B) Cells in an en-gal; UAS-dcr-1RNAi adult wing. The red line indicates the boundary between the anterior (a) dcr-1RNAi-non-expressing and posterior (p) dcr-1RNAi-expressing cells. Note the reduced cell size of the posterior cells. (C, D) Cuticle preparations of adult wings expressing GFP, dcr-1RNAi and/or dcr-1 in the patched (ptc, C) or engrailed (en, D) domains (labelled blue) in different genetic backgrounds. (E–G) Histograms plotting the size (E, F) and cell density values (G), normalized as a percent of the control GFP-expressing wing values, of the ptc and en domains expressing different transgenes. The error bars indicate the standard deviation. Only adult wing males were analysed. (E) In ptc>dcr-1RNAi, a significant decrease in the size of the ptc domain was observed when compared with ptc>GFP wings (P dcr-1RNAi, a significant decrease in the size of the en domain was observed when compared with en>GFP wings (P GFP wings (P<10−3). Coexpression of dcr-1-rescued tissue (P<10−10) and cell size (P GFP=100±8.4 (n of wings=10); ptc>dcr-1-RNAi=75±5.1 (n=12); en>GFP=100±5.7 (n=12); en> dcr-1RNAi=75±5.1 (n=10); en> dcr-1RNAi>dcr-1=110±5.8 (n=12). Cell density values: en>GFP=100±7.6 (n=10); en> dcr-1RNAi=123±9.4 (n=10); en> dcr-1RNAi>dcr-1=95±2.6 (n=10). (H, I) Adult wings with clones of cells lacking dcr-1 activity. The mutant tissue in adult wings (genotype: forked36a hs-FLP; FRT 82 P(forked+) M(3)95A2/FRT82 dcr-1Q1147X) was marked by absence of the P(forked+) rescue construct (see Materials and methods). In panel H, the red bar indicates the mutant bristles and blue arrows indicate the wild-type bristles. The red line in panel I indicates the boundary between wild-type and mutant tissue. Wild-type and M(3)95A2/+ adult wings show a similar cell size (Morata and Ripoll, 1975). Download figure Download PowerPoint To further study the requirement of Dcr-1 in tissue growth, we induced clones of dcr-1Q1147X mutant cells in the wing primordium and compared their size with their wild-type twin clones resulting from the same mitotic recombination event. In clones analysed 72 h after induction, activity of Dcr-1 was already reduced, monitored by the reduced activity of the bantam miRNA (Supplementary Figure S1). Clones of dcr-1Q1147X mutant cells were on average smaller than their corresponding wild-type twin clones (Figure 2B and C; see also references Friggi-Grelin et al, 2008; Martin et al, 2009). Clones of cells mutant for dcr-1d102, another allele of dcr-1, gave similar results (Figure 2A and C). We noticed that these clones were frequently fragmented (Figure 2D) and that many clones were eliminated from the wing disc by 96 h after induction (Figure 2E). A total of 31.5% of the wild-type twin clones induced 96 h before dissection were lacking their corresponding dcr-1 mutant clones (n of twin clones/wing disc=4.1; n of dcr-1 clones/wing disc=2.4; n of discs=10). Most dcr-1 mutant clones were positive for TUNEL staining, a marker of apoptotic cells (Figure 2F). These observations suggest that dcr-1 mutant cells are eliminated through cell competition, a process by which slower growing cells are detected and removed through apoptosis (Morata and Ripoll, 1975; Moreno et al, 2002; de la Cova et al, 2004; Moreno and Basler, 2004; Li and Baker, 2007). To test this we gave the mutant cells a relative growth advantage using the Minute technique to impair growth of the other cells (see section Materials and methods). In a Minute/+ background, dcr-1 mutant clones were recovered at the same frequency as wild-type control clones induced 96 h before dissection ([dcr-1 M(+)]=3.2 clones/wing disc, n=32 clones; [M(+)]=3 clones/wing disc, n=30 clones). However, dcr-1 mutant clones were much smaller than the wild-type control clones (Figure 2G and H). Similar results were obtained with clones of cells mutant for Argonaute-1, a component of the miRISC complex. Argonaute-1 mutant clones were smaller than their wild-type twin clones, were lost from the epithelium and their recovery rate was increased when given a growth advantage with the Minute technique (Supplementary Figure S1, and data not shown). Taken together, these results suggest that the miRNA machinery is required for cell and tissue growth. Figure 2.Dcr-1 and cell competition. (A, B) Wing discs with clones of cells lacking dcr-1 activity marked by absence of GFP (white). Clones were induced 72 h before dissection. Note the reduced size of the mutant clones (in black) when compared with the control wild-type twins (in white). (C) Graphs showing the relative sizes (clone areas, in arbitrary units) of individual pairs of dcr-1−/− clones (black bars) and dcr-1 +/+ twins (grey bars). Two different alleles of dcr-1 were used in panels A–C. Genotypes: hs-FLP; FRT 82 Ubi-GFP/FRT82 dcr-1Q1147X and hs-FLP; FRT 82 Ubi-GFP/FRT82 dcr-1d102. Only those wing discs with low frequency of clones and twins were scored to facilitate the quantification and reduce the possibility of fusion of neighbouring clones or twins. (D–F) Wing discs with clones of cells lacking dcr-1 activity marked by absence of GFP (white in panels D and E, green in panel F) and induced 72 h (D, F) or 96 h (E) before dissection. Note that mutant clones tend to break (red arrowheads in panel D) and enter apoptosis (labelled by TUNEL staining, red, F) 72 h after induction, and they are frequently lost from the epithelium 96 h after induction (E). (G) Wing discs with clones of cells lacking dcr-1 activity (right panels) or wild type for dcr-1 (left panels) and generated by the Minute technique to give clone cells a growth advantage. Clones were labelled by absence of GFP expression (white) and induced 96 h before dissection. The genotypes were: hs-FLP; FRT 82 Ubi-GFP M(3)95A2/FRT82 dcr-1Q1147X and hs-FLP; FRT 82 Ubi-GFP M(3)95A2/FRT82. (H) Histogram plotting the size of dcr-1 (n clones=21) and wild-type Minute(+) clones (n clones=18) induced 96 h before dissection. Clone size was normalized as a percent of the wild-type Minute (+) clone size. The error bars indicate the standard deviation. The difference between both genotypes was statistically significant (P dcr-1RNAi, a significant decrease in the size of the en domain was observed when compared with en>GFP wings (P<10−7 in males and P<10−3 in females). Halving the dose of dap or Rbf or coexpression of CycE significantly rescued this phenotype (P(dap4)<10−5, P(Rbfsls5)<10−5, P(CycE) dcr-1RNAi males, a significant increase in the cell density of the en domain was observed when compared with en>GFP wings (P<10−3). Halving the dose of dap or coexpression of CycE significantly rescued the cell size defects (P(dap4)<10−3, P(CycE) GFP=100±5.7 (n=12); en>dcr-1RNAi=75±5.1 (n=10); en>dcr-1RNAi; dap4/+=122±7.3 (n=7); en>dcr-1RNAi>CycE=111±4.5 (n=12). Tissue size values (females): en>GFP=100±7.4 (n=12); en>dcr-1RNAi=66±1.7 (n=12); Rbfsls5/+; en>dcr-1RNAi=80±3.1 (n=12). Cell density values (males): en>GFP=100±7.6 (n=10); en>dcr-1RNAi=123±9.4 (n=10); en>dcr-1RNAi; dap4/+=105±4.5 (n=10); en>dcr-1RNAi>CycE=106±4.5 (n=10). Download figure Download PowerPoint The G1–S transition is negatively regulated by the CDK inhibitor Dacapo (Dap, the Drosophila p21/p27 orthologue), which traps the CycE–CDK2 complex in a stable but inactive form (de Nooij et al, 1996; Lane et al, 1996), and by retinoblastoma family proteins Rbf1 and Rbf2, which interact with and negatively regulate E2F (van den Heuvel and Dyson, 2008). In cells with reduced Dcr-1 activity, Dap protein expression was increased (Figure 3I and J) and E2F activity was reduced (Figure 3E–H), visualized using the dE2F1-responsive reporter ORC1-GFP (Asano and Wharton, 1999; labelled dE2F in the figures) and PCNA, a target of dE2F. Wild-type control clones generated in a Minute background did not show any change in PCNA protein levels (Supplementary Figure S2). Altogether, these results suggest that miRNAs normally promote cell division by limiting the expression of Dap and by increasing the activity of E2F. Cell-cycle regulators like Dap, Rbf or CycE have been reported to not exert a direct effect on tissue growth in mature wing discs and adult wings (Neufeld et al, 1998; see also Supplementary Table S1). However, their expression is altered under conditions where reduced miRNA pathway activity affects tissue growth. In this context we asked whether manipulating CycE, Dap or Rbf levels would influence the growth-reducing effects of miRNA depletion. Increased CycE or reduced Dap or Rbf protein levels were able to overcome the effects of dcr-1 depletion. Under these circumstances, cell size was rescued (Figure 3M) and reduction in the size of the wing territory was compensated (Figure 3K and L). These results imply that the influence of cell-cycle regulators on tissue growth is not normally limiting, but that it can be shown under conditions where requirement for their activity is sensitized. Consistent with this view, increased CycE was shown to overcome the defects on tissue growth caused by depletion of Notch signalling in early wing and eye primordia (Kenyon et al, 2003; Rafel and Milan, 2008). Regulation of dMyc levels by the activity of Dcr-1 The proto-oncogene dMyc regulates cell growth, tissue growth and G1–S transition, and differences in dMyc levels induce cell competition in Drosophila tissues (Johnston et al, 1999; de la Cova et al, 2004; Moreno and Basler, 2004). Reduced dMyc expression leads to cellular and clonal phenotypes resembling some aspects of reduced Dcr-1 activity. Intriguingly, we found that dMyc protein and mRNA were expressed at lower than normal levels in cells depleted of dcr-1 (Figure 4A–F). We also monitored the effects of Dcr-1 depletion on the activity of the PI3K and hippo pathways, two pathways involved in growth control in Drosophila tissues (Neufeld, 2003; Pan, 2007), but found no changes (Supplementary Figure S3). These results suggest a specific role of the miRNA pathway in regulating dMyc protein levels. Figure 4.Dcr-1 regulates dMyc by repressing Mei-P26 protein levels. (A–F) Wild-type wing discs (E, F) or wing discs with reduced dcr-1 activity (A–D) labelled to visualize dMyc protein (red or white; A, B, D, E) or mRNA (purple; C, F) expression. In panel A, clones of cells were generated (genotype: hs-FLP; FRT 82 Ubi-GFP M(3)95A2/FRT82 dcr-1Q1147X) and marked by absence of GFP (green). In panels B–D, dcr-1RNAi was expressed in the patched (ptc, red arrowheads in panels B and C) or engrailed (en, red brackets in panel D) domains. The en domain was also labelled in panel D by expression of GFP. (G–I) Wing discs overexpressing mei-P26 (G, G′, H), dMyc and mei-P26 (I, J) or dMyc and GFP (K, L) in the engrailed (en; brackets in panels G, G′, H, I) domain and labelled to visualize dMyc protein (red or white; G, G′, I, K), Mei-P26 protein (green; I), GFP protein (green; K) or dMyc mRNA (purple; H, J, L). In panel G′, wing discs were cultured in the presence of MG132, a proteasome inhibitor, for3 h. (M–O) Wing discs with reduced dcr-1 activity labelled to visualize Mei-P26 protein (red or white) or mei-P26 mRNA (purple) expression. In panels M and M′, clones of dcr-1 mutant cells were generated (genotype: (M) hs-FLP; FRT 82 lacZ M(3)95A2/FRT82 dcr-1Q1147X and (M′) hs-FLP; FRT 82 lacZ/FRT82 dcr-1Q1147X) and marked by absence of β-gal (green). In panels N and O, dcr-1RNAi was expressed in the patched (ptc; red arrowheads) domains. In panel O, sense and antisense RNA probes were used. (P, Q) Wing discs expressing dcr-1RNAi and mei-P26RNAi (ID number: 7553) in the patched (ptc, red arrowheads) domain and labelled to visualize dMyc (green or white) and Mei-P26 (red or white) protein expression in panel P, and dMyc mRNA (purple) expression in panel Q. (R, S) Wing discs with clones of AGO1 mutant cells marked by absence of GFP and labelled to visualize Mei-P26 (R) and dMyc (S) protein expression (red or white).Genotype: hs-FLP; FRT G13 Ubi-GFP/FRT13 AGO114 (R) and hs-FLP; FRT G13 Ubi-GFP M(2)l2/FRTG13 AGO114 (S). Download figure Download PowerPoint We next determined the mechanism used by the miRNA machinery to regulate dMyc protein levels. The murine TRIM-NHL protein TRIM32 has been reported to bind to, ubiquitinate and degrade c-Myc (Schwamborn et al, 2009). We tested whether the Drosophila TRIM32 orthologue Mei-P26, known to regulate cell growth and proliferation in stem cells (Neumuller et al, 2008), had a similar role in wing disc cells. Overexpression of Mei-P26 induced a strong reduction in dMyc protein levels and a milder reduction in dMyc mRNA levels (Figure 4G and H). To investigate whether regulation of dMyc protein levels by Mei-P26 is a consequence of reduced dMyc mRNA levels, we analysed the capacity of Mei-P26 to downregulate exogenously expressed dMyc. For this purpose, we compared dMyc protein and mRNA levels in wing discs overexpressing dMyc and GFP (in en-gal4; UAS-GFP, UAS-dMyc larvae; Figure 4K and L) or dMyc and Mei-P26 (in en-gal4; UAS-Mei-P26, UAS-dMyc larvae; Figure 4I and J). Mei-P26 reduced exogenously induced dMyc protein with no obvious effect on dMyc mRNA levels. To determine whether dMyc protein might be degraded by Mei-P26 in a proteasome-dependent manner, we incubated wing discs overexpressing Mei-P26 with the proteasome inhibitor MG-132. In drug-treated wing discs, dMyc protein degradation was prevented (Figure 4G′). In control solvent-treated discs, overexpression of Mei-P26 induced a strong reduction in dMyc protein levels (data not shown). In wild-type wing discs, expression of dMyc is reduced along the dorsal–ventral (DV) compartment boundary by the activity of Notch (Herranz et al, 2008). In drug-treated wing discs overexpressing Mei-P26, we noticed that cells located at the DV boundary and expressing Mei-P26 expressed high levels of dMyc, suggesting that high levels of Mei-P26 override the effect of Notch on dMyc levels. Consistent with this view, the Notch-regulated gene wingless was also repressed upon Mei-P26 overexpression (Supplementary Figure S4). We next analysed dMyc protein levels in a situation of reduced Mei-P26 activity. As the available mei-P26 alleles did not lead to a visible reduction in Mei-P26 protein levels in the clones of cells in the wing disc (data not shown), we used the Gal4/UAS technique to express three independent RNAi transgenes that target mei-P26 (mei-P26RNAi−7553, mei-P26RNAi−106754 and mei-P26RNAi−11429). All three were able to visibly reduce Mei-P26 protein levels when expressed in specific territories in the wing disc (Figure 4P; Supplementary Figure S4, and data not shown). Expression of these constructs did not induce a significant change in dMyc protein levels in the developing wing (Figure 4P and Supplementary Figure S4). These data suggest that ‘normal’ levels of Mei-P26 activity are not limiting for dMyc protein levels. However as shown above, elevated Mei-P26 levels can lead to reduction of dMyc. This raises the possibility that regulation of Mei-P26 activity could provide a means to control dMyc levels. Consistent with this view, mei-P26 is predicted to be a target for regulation by miRNAs (Stark et al, 2005) and cells mutant for dcr-1 or with reduced dcr-1 activity showed increased levels of Mei-P26 protein but not mei-P26 mRNA (Figure 4M–O). Cells depleted of Argonaute-1, a component of the miRISC complex, also expressed higher levels of Mei-P26 and showed reduced levels of dMyc (Figure 4R and S). Archipelago, the F-box component of an SCF-ubiquitin ligase, is known to negatively regulate the levels and activity of dMyc protein in vivo (Moberg et al, 2004). Archipelago protein levels were not affected in cells mutant for dcr-1 (Supplementary Figure S3). To test whether the increase in Mei-P26 protein levels was responsible for the reduction in dMyc expression in dcr-1 mutant cells, we analysed dMyc protein and mRNA levels when both Dcr-1 and Mei-P26 were depleted. Expression of mei-P26RNAi together with dcr-1RNAi was able to prevent the increase of Mei-P26 and thereby prevent the reduction of dMyc protein and mRNA levels that was seen when Dcr-1 was depleted alone (Figure 4P and Q, and Supplementary Figure S4). These results indicate that the miRNA machinery regulates dMyc by repressing Mei-P26 protein production. Contribution of dMyc to the growth and E2F activity defects caused by Dcr-1 depletion The above results indicate that the miRNA machinery regulates dMyc levels through the activity of Mei-P26 (Figure 4). As reduced Dcr-1 activity led to cellular and clonal phenotypes resembling the consequences of reduced dMyc activity (Johnston et al, 1999; de la Cova et al, 2004; Moreno and Basler, 2004), we examined the contribution of dMyc to the defects caused by depletion of Dcr-1. For this purpose, we restored dMyc expression and analysed tissue and cell size in dcr-1RNAi-expressing adult wings. The Gal4/UAS technique was used to express high levels of dMyc in the same domain as dcr-1RNAi. As well, two different heterologous promoters (heat-shock, hs, and tubulin, tub) were used to drive dMyc expression throughout the wing disc (hs-dMyc (Johnston et al, 1999) and tub-dMyc (Moreno and Basler, 2004)). The tissue and cell size defects caused by dcr-1RNAi in adult wings were largely rescued in all three combinations (Figure 5A). Notably, hs-dMyc does not express dMyc at levels that cause significant tissue overgrowth on its own under the conditions used (Supplementary Table S1). Therefore it is unlikely that suppression of the Dcr-1 depletion phenotypes by hs-dMyc reflects the independent action of Dcr-1 depletion and dMyc overexpression. This possibility cannot be excluded for the tub-dMyc and UAS-dMyc combinations, which express higher levels of dMyc. To further investigate this issue, we made use of a different means to restore dMyc activity that does not involve dMyc overexpression.