The miRNA machinery regulates dMyc protein levels by targeting Mei-P26. An illustration describing the regulatory network described in this work by which the miRNA machinery maintains dMyc levels by targeting Mei-P26.

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-1^Q1147X) and marked by absence of GFP (green). In panels B–D, dcr-1^RNAi 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-1^Q1147X and (M′) hs-FLP; FRT 82 lacZ/FRT82 dcr-1^Q1147X) and marked by absence of β-gal (green). In panels N and O, dcr-1^RNAi 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-1^RNAi and mei-P26^RNAi (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).

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-1^Q1147X and hs-FLP; FRT 82 Ubi-GFP/FRT82 dcr-1^d102. 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-1^Q1147X 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<10⁻⁸).

Dcr-1 regulates G₁–S transition. (A–J) Wing discs with clones of cells lacking dcr-1 activity (A, D, E, G, I; genotype: hs-FLP; FRT 82 lacZ M(3)95A2/FRT82 dcr-1^Q1147X) marked by absence of β-gal (green), or expressing dcr-1^RNAi (B, C, F, H, J) in the patched (ptc) domain (red arrowheads) and labelled to visualize in pink or white cyclin-B (CycB; A, B), cyclin-E (CycE; C, D), dE2F activity (an E2F1 responsive reporter ORC1-GFP was used, antibody to GFP; E, F), PCNA (G, H) and Dacapo (Dap; I, J) protein expression. Clones were induced 72 h before dissection. (K) Cuticle preparations of adult wings expressing dcr-1^RNAi in the engrailed (en) domain in several genetic backgrounds. (L, M) Histograms plotting the size (L) and cell density (M) of the en domain in adult wings expressing dcr-1^RNAi in different genetic backgrounds. Tissue size and cell density values were normalized as a percent of the values of control GFP-expressing wings. The error bars indicate the standard deviation. Adult wing males and females were analysed. In en>dcr-1^RNAi, 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(Rbf^sls5)<10⁻⁵, P(CycE)<10⁻⁸). In en>dcr-1^RNAi 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) <10⁻³). Tissue size values (males): en>GFP=100±5.7 (n=12); en>dcr-1^RNAi=75±5.1 (n=10); en>dcr-1^RNAi; dap⁴/+=122±7.3 (n=7); en>dcr-1^RNAi>CycE=111±4.5 (n=12). Tissue size values (females): en>GFP=100±7.4 (n=12); en>dcr-1^RNAi=66±1.7 (n=12); Rbf^sls5/+; en>dcr-1^RNAi=80±3.1 (n=12). Cell density values (males): en>GFP=100±7.6 (n=10); en>dcr-1^RNAi=123±9.4 (n=10); en>dcr-1^RNAi; dap⁴/+=105±4.5 (n=10); en>dcr-1^RNAi>CycE=106±4.5 (n=10).

The role of bantam in mediating the activity of Dcr-1 in regulating Dap and Mei-P26 protein levels. (A) Histograms plotting the size and cell density values of the en domains expressing GFP or dcr-1^RNAi. Tissue size and density values were normalized as a percent of the values of control GFP-expressing wings. The error bars indicate the standard deviation. Only adult wing males were analysed. In en>dcr-1^RNAi, a significant decrease in the size of the en domain and a significant increase in the cell density was observed when compared with en>GFP wings (P<10⁻⁷ and P<10⁻³, respectively). Mild ubiquitous expression of bantam (with the hs-bantam transgene) was able to rescue tissue size (P<10⁻⁹) and cell density (P<10⁻⁹). Tissue size values: en>GFP=100±5.7 (n=12); en>dcr-1^RNAi=75±5.1 (n=10); en>dcr-1^RNAi; hs-bantam=119±6.3 (n=12). Cell density values: en>GFP=100±7.6 (n=10); en>dcr-1^RNAi=123±9.4 (n=10); en>dcr-1^RNAi; hs-bantam=94±4.2 (n=10). (B–E) Wing discs labelled to visualize Mei-P26 (red or white; B, D, E) or dMyc (red or white; C), and GFP protein expression in ptc-Gal4; UAS-dcr-1^RNAi, UAS-bantam-GFP (B, C) and ptc-Gal4; UAS-bantam-GFP (D) larvae, and in clones of cells mutant for bantam (genotype: hs-FLP; M(3L) ubi-GFP FRT80B/ban^Δ1FRT80B) and marked by the absence of GFP (green) (E). The patched (ptc) domain is indicated by a red arrowhead. (F) Gal4-directed expression of UAS-bantam inhibited luciferase activity from a reporter containing the mei-P26 3′UTR (see Materials and methods), and in a much weaker manner the mutant 3′UTR in which the bantam 5′ seed region was deleted. The error bars indicate the standard deviation. The difference between intact and mutated UTR reporters was statistically significant (P<0.05). (G) Cartoon depicting the different Firefly-luciferase constructs containing wild-type or mutated mei-P26 3′UTR used in panel F. (H) Expression of mir-137 genomic fragments inhibited luciferase activity from a reporter containing the mei-P26 3′UTR (see Materials and methods), and in a much weaker manner the mutant 3′UTRs (mut1 and mut2) in which the mir-137 target sites were mutated. The error bars indicate the standard deviation. The difference between intact and mutated UTR reporters was statistically significant (P<0.05). (I) Cartoon depicting the different Firefly-luciferase constructs containing wild-type or mutated forms of the mei-P26 3′UTR used in panel H. The seed region is marked in red and the mutation are marked in blue and underlined.

dMyc contributes to the activity of Dcr-1 in regulating cell and tissue growth and E2F activity. (A) Histograms plotting the size and cell density values of the en domains expressing different transgenes. Tissue size and density values were normalized as a percent of the values of control GFP-expressing wings. The error bars indicate the standard deviation. Only adult wing males were analysed. In en>dcr-1^RNAi, a significant decrease in the size of the en domain was observed when compared with en>GFP wings (P<10⁻⁷). Coexpression of dMyc or mei-P26^RNAi significantly rescued this phenotype (P(dMyc)<10⁻⁹ and P(mei-P26^RNAi)<10⁻⁴). Mild ubiquitous expression of dMyc was also able to rescue this phenotype (P(hs-dMyc)<10⁻⁵ and P(tub-dMyc)<10⁻⁹). In en>dcr-1^RNAi, a significant increase in the cell density of the en domain was observed when compared with en>GFP wings (P<10⁻³). Coexpression of dMyc or mei-P26^RNAi significantly rescued the cell size defects (P(dMyc) <10⁻⁵ and P(mei-P26^RNAi)<10⁻⁴). Mild ubiquitous expression of dMyc was also able to rescue this phenotype (P(hs-dMyc)<10⁻⁸ and P(tub-dMyc)<0,08). Tissue size values: en>GFP=100±5.7 (n=12); en> dcr-1^RNAi=75±5.1 (n=10); en> dcr-1^RNAi>dMyc=118±9.5 (n=12); en>dcr-1^RNAi; hs-dMyc=99±13.3 (n=10); en>dcr-1^RNAi; tub-dMyc=108±5.8 (n=10); en>dcr-1^RNAi;mei-P26^{RNAi 106 754}=86±11.2 (n=10). Cell density values: en>GFP=100±7.6 (n=10); en>dcr-1^RNAi=123±9.4 (n=10); en>dcr-1^RNAi>dMyc=105±2.98 (n=10); en>dcr-1^RNAi; hs-dMyc=89±3.5 (n=10); en>dcr-1^RNAi; tub-dMyc=111±17 (n=10); en>dcr-1^RNAi;mei-P26^{RNAi 106 754}=105±3 (n=10). (B–D) Wing discs with clones of wild-type cells (B), mutant cells for dcr-1 (C) and mutant cells for dcr-1 and expressing dMyc (D) (genotypes: A: hs-FLP tub-Gal4 UAS-GFP; FRT82 arm-lacZ/FRT82 Gal80; B: hs-FLP tub-Gal4 UAS-GFP; FRT82 dcr^Q1147X/FRT82 Gal80; C: hs-FLP tub-Gal4 UAS-GFP; UAS-dMyc/+; FRT82 dcr^Q1147X/FRT82 Gal80). Clones were labelled by presence of GFP and induced 96 h before dissection. In panel B, twin clones were labelled by absence of β-gal expression (in red). Wing discs in panels C and D were labelled to visualize dMyc protein expression in red. (E) Frequency and size (in number of cells) of clones shown in panels B–D. To minimize the effects of fusion of clones, only those wild-type clones with one twin were quantified and wing discs with obvious fusions were discarded. (F–R) Wing discs labelled to visualize in red or white cyclin-B (CycB; F, K, O), PCNA (G, L, P), cyclin-E (CycE; H, M, Q) and Dacapo (Dap; I, J, N, R) protein expression, and in green GFP protein expression. In panels F–I, clones of cells mutant for dcr-1 and expressing GFP and dMyc were generated (genotype: hs-FLP tub-Gal4 UAS-GFP; UAS-dMyc/+; FRT82 dcr^Q1147X/FRT82 Gal80). Clones were induced 72 h before dissection. In panels J–R, the following transgenes were expressed in the patched (ptc) domain: dcr-1^RNAi, dMyc and GFP (J), dcr-1^RNAi and GFP (K–N) and dcr-1^RNAi and mei-P26^RNAi (ID number: 106754, O-R). In panels K–N, dMyc was expressed at lower levels using the tub-dMyc construct.

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-1^RNAiUAS-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 dicer^RNAi 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-1^RNAi adult wing. The red line indicates the boundary between the anterior (a) dcr-1^RNAi-non-expressing and posterior (p) dcr-1^RNAi-expressing cells. Note the reduced cell size of the posterior cells. (C, D) Cuticle preparations of adult wings expressing GFP, dcr-1^RNAi 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-1^RNAi, a significant decrease in the size of the ptc domain was observed when compared with ptc>GFP wings (P<10⁻⁵). (F, G) In en>dcr-1^RNAi, a significant decrease in the size of the en domain was observed when compared with en>GFP wings (P<10⁻⁷), and a significant increase in the cell density of the en domain was observed when compared with en>GFP wings (P<10⁻³). Coexpression of dcr-1-rescued tissue (P<10⁻¹⁰) and cell size (P<10⁻²) defects. Tissue size values: ptc>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-1^RNAi=75±5.1 (n=10); en> dcr-1^RNAi>dcr-1=110±5.8 (n=12). Cell density values: en>GFP=100±7.6 (n=10); en> dcr-1^RNAi=123±9.4 (n=10); en> dcr-1^RNAi>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: forked^36a hs-FLP; FRT 82 P(forked+) M(3)95A2/FRT82 dcr-1^Q1147X) 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).