miR-24 inhibits cellular proliferation by increasing the G1 compartment

Since cessation of cell proliferation is a hallmark of terminal differentiation, we first examined whether proliferation is altered by either inhibiting or enhancing miR-24 function by transfecting cells with miR-24 2′-OMe antisense oligonucleotide (ASO) or miRNA mimics, respectively. When K562 cells were transfected with miR-24 ASO, miR-24 was dramatically and specifically reduced by qRT-PCR 36 hr later (Fig. 1B). DNA replication, measured by thymidine incorporation, doubled in cells transfected with miR-24 ASO compared to cells transfected with control ASO (Fig. 1C). When K562 cells were differentiated with TPA for 4 hr, thymidine incorporation declined by 60%. However, in cells transfected with miR-24 ASO and treated with TPA, thymidine uptake was indistinguishable from that of the control ASO-transfected, but TPA untreated, cells (Fig. 1C). Therefore, miR-24 ASO fully restored proliferation to differentiating K562 cells. To examine whether miR-24 also inhibits cell proliferation in nontransformed cells, we next antagonized miR-24 in early passage WI-38 and IMR-90 normal diploid fibroblasts. Antagonizing miR-24 in WI-38 and IMR-90 cells dramatically reduced miR-24 (Fig. 1D) and increased thymidine uptake >2-fold 48 hr after transfection (Fig. 1E). Conversely, over-expressing miR-24 in HepG2 cells synchronized with nocodazole, which typically leads to mitotic arrest and only ~8% of cells in G1, increased G1 cells 3-fold (22%; miR-24 vs cel-miR-67, p<0.001) (Fig. 1F).

We next analyzed how miR-24 expression changes during normal cell cycle progression using K562 cells released at various times from nocodazole treatment, which synchronized them in G2/M (Bar-Joseph et al., 2008; O'Donnell et al., 2005) (Fig. 1G,H). Before release, 90% of cells were in G2/M; 8 hr later 65% were in G1 and 12 hr after removing nocodazole, 45% were in S phase. miR-24 was low in G2/M, increased >3-fold by 8 hr when most cells were in G1 and then declined by 12 hr as cells progressed into S phase. These results suggest that miR-24 is most highly expressed in G1. Taken together with our finding that cells transduced with miR-24 mimics accumulate in G1, these results suggest that miR-24 regulates cell cycle progression mostly by blocking or delaying the G1/S transition.

Most genes down-regulated by miR-24 contain a miR-24 seed in their 3′UTR

We next sought to identify miR-24-regulated targets and cellular pathways by comparing mRNA microarrays of cells transfected with miR-24 or control miRNA (cel-miR-67) mimic. Transfecting HepG2 cells, which have low endogenous miR-24 levels (Fig. 2A), with a miR-24 mimic increased miR-24 expression ~80-fold compared with control cells (Fig. 2B). Total RNA, isolated from duplicate miRNA-transfected samples 48 hr later, was amplified, labeled and hybridized to Illumina mRNA microarrays. 248 mRNAs were down-regulated at least 2-fold by miR-24 over-expression (Z-ratio>1.5) (Suppl. Table 1). We validated the microarray data by performing qRT-PCR amplification for 9 randomly chosen down-regulated genes that spanned the range of significantly down-regulated genes (Z-ratios, 1.6-6.1), of which 6 (CNDP2, TOP1, PER2, MBD6, H2AFX, STX16) were predicted miR-24 targets by TargetScan 4.2 and 3 (UBD, BCL2L12, ZNF317) were not (Fig. 2C). H2AFX was previously shown to be directly regulated by miR-24 (Lal et al., 2009). All 9 genes were significantly down-regulated, and the extent of down-regulation correlated well with their reduced expression in the microarray, validating the quality of the microarray data.

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Fig. 2

mRNA down-regulation after miR-24 over-expression (A) HepG2 cells express low levels of miR-24, assayed by qRT-PCR analysis normalized to U6, compared to HeLa, WI-38, HL60 and K562 cells. (B) Effective increase in miR-24 in HepG2 cells 48 hr after transfection with miR-24 mimic compared to cel-miR-67-transfected control cells. #, p<0.005. (C) Genes identified by microarray as down-regulated by miR-24 over-expression were confirmed to be down-regulated by qRT-PCR normalized to GAPDH. UBC is a housekeeping gene. Cells were transfected with cel-miR-67 (black) or miR-24 mimic (white). **, p<0.01; #, p<0.005, ##, p<0.001. The down-regulated genes are graphed in order of their down-regulation on the microarray; the Z-ratio of the microarray analysis is shown below. Error bars represent SD from 3 independent experiments (A, B and C). (D) Venn diagram of genes down-regulated by miR-24 in HepG2 cells and genes predicted to be regulated by miR-24 using TargetScan 4.2. Of the 100 predicted genes, whose mRNA is also significantly down-regulated, only 20 have conserved predicted miR-24 recognition sites. TargetScan 4.2 predicts 349 conserved miR-24 targets and many more that are not conserved. (E) Sites complementary to the miR-24 seed are enriched in the 3′UTR of down-regulated transcripts. The table shows the frequency of perfect hexamer (positions 2-7), heptamer (positions 2-8) and octamer (positions 2-9) miR-24 3′UTR seeds in the down-regulated genes.

To determine what fraction of the down-regulated genes are likely direct targets of miR-24, we used two approaches. First, we compared our experimental list of down-regulated transcripts with TargetScan predictions. 100 down-regulated genes were also predicted by TargetScan 4.2 (Lewis et al., 2003) (Fig. 2D, Suppl. Table 1). Amongst these 100 targets, however, only 20 have predicted miR-24 3′UTR miRNA recognition sites (MRE) conserved in human, mouse, rat and dog. Second, we examined the frequency of a 3′UTR sequence perfectly complementary to the miR-24 seed (hexamer (positions 2-7), heptamer (positions 2-8) and octamer (positions 2-9)). The down-regulated genes were highly enriched for miR-24 seed matches (Fig. 2E, Suppl. Table 1). Just over half of the 219 miR-24 down-regulated transcripts that have an annotated 3′UTR contain a 3′UTR complementary hexamer sequence (53%, p=2×10^-16 relative to the background frequency of the seed in the known transcriptome), 32% have a heptamer match (p=7×10^-15) and 8% have an octamer seed match (p=0.0002). This significant enrichment of predicted miR-24 target genes and the high frequency of genes containing perfect seed matches suggest that a substantial proportion of the down-regulated genes may be direct miR-24 targets.

Cell cycle and DNA repair genes are regulated by miR-24

We next performed a gene ontology (GO) analysis (GeneGo, Inc) to identify cellular pathways enriched in the set of genes down-regulated after ectopic miR-24 expression. This analysis did not yield a tightly focused set of functions for miR-24 (Suppl. Table 2). Therefore, we next looked at whether the 100 down-regulated genes, which were also predicted miR-24 targets, are enriched for specific biological processes. A functional enrichment and network analysis revealed statistically significant enrichment for 49 processes, many of which are overlapping (Fig. 3A; Suppl. Table 3). The top 3 most enriched GO processes involve DNA repair (DNA damage checkpoint, double strand break repair by homologous recombination, and recombinational repair; each enriched with significance of p=0.0001). We previously found that miR-24 interferes with the DNA damage response in terminally differentiated hematopoietic cells, predominantly by reducing expression of the histone variant H2AFX, which recruits and retains DNA repair factors at double-strand breaks (Lal et al., 2009). In addition, multiple GO processes involved in cell cycle regulation were also highly enriched (regulation of cell cycle, p=0.0002; DNA integrity checkpoint p=0.0003; cell cycle arrest, p=0.0007; cell cycle, p=0.001; DNA recombination, p=0.001). This was not surprising based on the effect of miR-24 on cell cycle progression. When networks were developed to identify known directly interacting proteins from these over-represented biological processes, there was one cluster of 6 genes centered around MYC (c-myc) and three other small clusters involving 2 or 3 genes (Suppl. Fig. 2A); the other 87 genes in the data set lack any previously annotated direct interactions. Because the TargetScan algorithm might miss some important miRNA-regulated genes, we also constructed a direct interaction network from the 248 down-regulated mRNAs. The direct interaction network constructed from all significantly down-regulated mRNAs was a highly interactive set of 68 interacting genes, many of which are important in cell cycle regulation. The major connected network of miR-24 down-regulated genes is shown in Fig. 3B; there were also some smaller networks (Suppl. Fig. 2B). Key nodes of the major network are MYC (22 interactions), E2F2 (6), VHL (6), CDC2 (6), CCNB1 (5), and CDKN1B (5). The MYC and E2F2 transcription factors play a central role in regulating G1/S transition and progression through S. They inhibit cell differentiation and apoptosis and promote cellular transformation (Bracken et al., 2004; Lebofsky and Walter, 2007). MYC regulates the transcription of other genes in the network, including E2F2, CDKN1B, CCNB1, CDC2, CDCA7, and RRM2. E2F2 also regulates the transcription of other genes in the network, including CDC2, MYC, RRM2 and the minichromosome maintenance proteins MCM4 and MCM10 that are essential for initiating DNA replication. These analyses support our experimental findings that miR-24 regulates cell cycle progression and DNA repair.

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Fig. 3

Bioinformatic analysis of miR-24 down-regulated genes suggests that miR-24 regulates cell cycle progression and DNA repair (A) Top 15 over-represented cellular processes for the 100 overlapping genes in Fig. 2D. Over-represented processes are sorted by Score (-log [p-value]). A highly positive score suggests that the subnetwork is highly saturated with genes identified experimentally and doesn't have many nodes not experimentally identified. The complete list of 100 genes in the overlap and further statistical analysis is provided in Suppl. Table 1 and 3. The dotted line represents the statistically significant limit. (B) Major direct interaction network of the 248 genes significantly down-regulated after transfection of HepG2 cells with miR-24 mimics. Nodes with at least 5 interactions (including auto-regulation) are highlighted (symbols: transcription factor, ; enzyme, ; kinase, ; ligand-dependent nuclear receptor, ; transporter, ; phosphatase, ; peptidase, ; translation regulator, ; other, ●).

miR-24 regulates MYC by binding to its 3′UTR

To determine whether miR-24 directly regulates the down-regulated genes, we began with MYC, since it is a key node of the interaction network, plays an important role in cell cycle progression and is a predicted miR-24 target. We transfected HepG2 and K562 cells with miR-24 mimics and 48 hr later, measured MYC mRNA by qRT-PCR. miR-24 over-expression decreased MYC mRNA by ~2-4 fold relative to GAPDH in HepG2 and K562 cells (Fig. 4A,B). UBC mRNA, a control gene, did not change significantly. MYC protein also decreased substantially (85%) (Fig. 4C). To investigate whether reduced MYC expression was directly mediated by miR-24, we measured changes after miR-24 co-transfection in luciferase activity from a MYC 3′UTR reporter. miR-24 reduced luciferase activity 2.2-fold (Fig. 4D). We next sought to identify miR-24 MREs in the MYC 3′UTR. TargetScan 4.2 predicts a single MRE containing a poorly conserved 7-mer exact seed match at positions 462-468 (although the recent TargetScan 5.0 algorithm does not list MYC as a miR-24 target), while rna22, an algorithm that does not require a seed match (Miranda et al., 2006), identifies 6 potential miR-24 MREs in the 488 nt MYC 3′UTR, including the TargetScan 4.2-predicted MRE (MRE6) (Fig. 4E). miR-24 over-expression specifically and significantly reduced luciferase activity by 1.9- and 3.9-fold for MRE3 and MRE6, respectively, but the other MREs had no significant effect on luciferase activity (Fig. 4F). MRE3 has no seed, but has extensive complementarity with the 3′end of miR-24. Point mutations of MRE3 and MRE6 that disrupted miR-24 binding restored luciferase activity (Fig. 4G,H). These findings suggest that miR-24 binds to 2 partially complementary sites (MRE3 and MRE6) in the MYC 3′UTR.

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Fig. 4

miR-24 regulates MYC expression. miR-24 over-expression in HepG2 (A) or K562 cells (B) decreases MYC mRNA, analyzed by qRT-PCR and normalized to GAPDH (black, cel-miR-67; white, miR-24). (C) MYC protein is decreased in K562 cells upon miR-24 over-expression. Densitometry was used to quantify protein; α-tubulin served as loading control. (D) miR-24 targets the MYC 3′UTR in a luciferase reporter assay. HepG2 cells were transfected with control miRNA (black) or miR-24 (white) mimic for 48 hr and then with MYC 3′UTR-luciferase reporter (MYC) or vector (V) for 24 hr. (E) Predicted binding sites in the MYC 3′UTR for miR-24 (MRE1-6) by rna22. The numbers in parenthesis correspond to the position in the MYC 3′UTR. Perfect matches are indicated by a line; G:U pairs by :. (F-H) miR-24 regulates MRE3 and MRE6 by luciferase reporter assay. HepG2 cells were co-transfected with cel-miR-67 (black) or miR-24 (white) mimics and luciferase reporters containing the wild type (wt) MRE1-6 in (E,F) or mutated (mt) MRE3 and MRE6 in (G,H) or vector (V). Luciferase activity was measured 48 hr after transfection. In (G), red letters denote point mutations that disrupt base pairing. Mean ± SD, normalized to vector control, of 3 independent experiments is shown. *, p<0.05; **, p< 0.01.

miR-24 down-modulates E2F2

We next examined the effect of miR-24 on E2F2, since E2F2 is down-regulated by miR-24 over-expression by microarray, is a key node in the gene interaction network (Fig. 3B), and plays a crucial role in regulating progression through G1, where miR-24-over-expressing cells pile up (Polager and Ginsberg, 2006). qRT-PCR analysis confirmed that E2F2 mRNA was significantly down-regulated by over-expressing miR-24 (Fig. 5A). In addition, the related E2F family members, E2F1 and E2F3, were also significantly decreased, although these two genes were not identified by the less sensitive microarray analysis. E2F1 and E2F3 down-regulation may be secondary to E2F2 down-regulation since the E2F-family of transcription factors regulate each other (Bracken et al., 2004; Vernell et al., 2003) or may be mediated by MYC, since MYC and E2F1 have been shown to transactivate each other (Fernandez et al., 2003). As expected, E2F2 protein (Fig. 5B) was also substantially reduced (9-fold).

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Fig. 5

miR-24 over-expression downregulates E2Fs and some E2F-regulated genes (A) miR-24 significantly reduces mRNA levels of E2F1, E2F2 and E2F3 and of some E2F-target genes (RRM2, CHEK1, CCNA2, FEN1, MCM4, MCM10, CDC2, AURKB), but not BRCA1 and PCNA. CDK4, a key MYC target gene, is also down-regulated. HepG2 cells were transfected with miR-24 (white) or control mimics (black) for 48 hrs, and E2F and their target mRNAs were measured by qRT-PCR. Data normalized to GAPDH are expressed relative to control mimic-transfected cells. UBC is a control housekeeping gene. The Z-ratios refer to the significantly down-regulated mRNAs in the mRNA microarray in miR-24-over-expressing cells. (B) Protein expression of miR-24 target genes is substantially reduced in miR-24 mimic-transfected K562 cells 72 hr after transfection, relative to control miRNA-transfected cells. Densitometry was used to quantify protein relative to α-tubulin. HuR and α-tubulin are loading controls. (C) miR-24 levels significantly increased when K562 cells were transfected with 10 or 50 nM miR-24 mimics as measured by qRT-PCR analysis. Expression relative to U6 snRNA is depicted normalized to control cells. (D) A 4-fold increase in miR-24, obtained by transfecting 10 nM miR-24 mimic, reduces target proteins. Cell lysates of K562 cells, obtained 72 hr after transfection with indicated concentrations of control or miR-24 mimics, were analyzed by immunoblot. α-tubulin and HuR are loading controls. Error bars represent mean ± SD from 3 independent experiments. *, p<0.05, **, p<0.01; #, p<0.005.

miR-24 down-regulates multiple E2F- and MYC-regulated genes

The E2F transcription factors activate the transcription of many genes essential for DNA replication, cell cycle progression and DNA repair. If miR-24 over-expression down-regulates E2F2, E2F2 target gene mRNAs would also be expected to decline after ectopic miR-24 expression. The effect of ectopic miR-24 expression in HepG2 cells on transcripts of 10 E2F targets important for cell cycle progression and DNA repair (AURKB, BRCA1, CCNA2, CDC2, CHEK1, FEN1, PCNA, RRM2, MCM4, MCM10) was analyzed 48 hr later. Eight of the 10 transcripts, with the exception of BRCA1 and PCNA, were significantly reduced (>40%) (Fig. 5A). A subset of these genes (CDC2, MCM4, MCM10, RRM2 and FEN1) was also significantly down-regulated by miR-24 over-expression by microarray (Suppl. Table 1). mRNA microarray may not be sensitive enough to identify some genes whose expression is suppressed, either directly or indirectly, by a miRNA. Protein levels of E2F2 and all 7 miR-24 target genes examined (AURKB, BRCA1, CCNA2, CDC2, CHEK1, FEN1, PCNA), quantified by densitometry of immunoblots, decreased by at least 2-fold (Fig. 5B). A possible explanation for the lack of correlation between the mRNA and protein levels of BRCA1 and PCNA is that mRNA levels were measured 48 hr after transfection of one cell type (HepG2) while protein levels were assayed 72 hr post-transfection in K562 cells. In fact, BRCA1, but not PCNA, mRNA was reduced by ~2-fold when K562 cells were transfected with miR-24 for 3 d (Suppl. Fig. 5).

Since miR-24 over-expression reduced MYC protein by 85% (Fig. 4C), MYC target genes should also be down-regulated. Consistent with this hypothesis, 11 known MYC-regulated genes (ATAD3A, ACTL6A, ARHGEF7, CCNB1, CDCA7, EXOSC8, E2F2, METAP2, N-PAC, RRM2 and UBE2C) were significantly down-regulated in miR-24 over-expressing HepG2 cells by mRNA microarray (Suppl. Table 1). Among MYC-regulated genes, CDK4 is an important mediator of MYC's effects on cellular proliferation (Hermeking et al., 2000). Although CDK4 mRNA was not significantly altered by microarray, CDK4 mRNA declined ~4-fold after ectopic expression of miR-24 in HepG2 cells by more sensitive qRT-PCR assay (Fig. 5A) and CDK4 protein became undetectable (Fig. 5B). Therefore, miR-24 over-expression decreases the levels of many genes important in cell cycle progression.

In these experiments, we over-expressed miR-24 ~80-fold above the level in undifferentiated K562 cells, whereas the physiological increase after TPA treatment of K562 cells is only 8-fold. To determine whether these genes are regulated by a physiological increase in miR-24, these experiments were repeated by transfecting K562 cells with varying miR-24 mimic concentrations (2-50 nM). Transfection of 2 nM miR-24 did not significantly alter miR-24, while 10 and 50 nM miR-24 increased miR-24 levels by 4- and 28-fold, respectively (Fig 5C). E2F2, MYC and 3 of 4 other E2F2-regulated mRNAs (AURKB, CCNA2, H2AX, but not PCNA) (Suppl. Fig. 5) and protein levels of all 7 genes tested (E2F2, AURKB, CHEK1, CCNA2, CDK4, MYC and PCNA) were all significantly reduced by a 4-fold increase in miR-24 (Fig. 5D). Therefore the genes identified by mRNA microarray down-regulated after ectopic miR-24 expression, are likely physiologically relevant direct and/or indirect miR-24 targets.

E2F2 and some E2F-target genes are directly regulated by “seedless” 3′UTR MREs

None of the 3 E2F paralogs (E2F1, E2F2 and E2F3), is a predicted target of miR-24 and their 3′UTRs do not contain a miR-24 seed match sequence. We nonetheless tested whether the E2F 3′UTRs might be directly regulated by miR-24 by luciferase assay. The E2F2, but not E2F1 or E2F3, 3′UTR significantly repressed luciferase activity in a miR-24-dependent manner (Fig. 6A) suggesting that E2F2 is a direct miR-24 target. rna22 identified 5 candidate E2F2 3′UTR miR-24 MREs (Fig. 6B, Suppl. Fig. 3). miR-24 significantly suppressed luciferase activity of a reporter gene containing the E2F2 MRE1. E2F2 MRE1 does not have a seed match in the 3′UTR, even if G:U wobbles are allowed, but has extensive complementarity to miR-24 elsewhere. Point mutations that disrupt base pairing between miR-24 and E2F2 MRE1 rescued luciferase expression, verifying that miR-24 specifically recognizes the E2F2 MRE1.

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Fig. 6

E2F2 and multiple E2F-target genes are direct targets of miR-24, recognized by “seedless” MREs. (A) miR-24 silences the expression of luciferase genes engineered with the 3′UTR of E2F2, but not with E2F1 or E2F3 3′UTRs, suggesting that E2F2 is a direct miR-24 target but E2F1 and E2F3 are down-regulated indirectly. Luciferase assays were performed in HepG2 cells over-expressing miR-24 (white) or control mimics (black). (B) miR-24 down-regulates luciferase activity of a reporter gene containing wild-type (wt) E2F2 MRE1. Mutations in the miR-24 pairing residues (mt) rescue luciferase expression (sequences and luciferase assays for candidate E2F2 wt 3′UTR MREs are shown in Suppl. Fig. 3). (C) miR-24 targets the 3′UTR of E2F-regulated genes (AURKB, BRCA1, CCNA2, CDC2, FEN1) and CDK4, a MYC-regulated gene. CHEK1 and PCNA 3′UTRs are not regulated by miR-24. HepG2 cells were co-transfected with a luciferase reporter containing the 3′UTR of the indicated gene and control miRNA (black) or synthetic miR-24 (white) for 48 hr. Expression of the unmodified luciferase vector (V) is unchanged by miR-24. (D) Predicted binding sites in the 3′UTR of genes whose 3′UTR was repressed by miR-24 in (C) and binding site mutations tested (indicated in red). (E) Expression of reporter genes containing wild-type (wt) AURKB MRE1, BRCA1 MRE5, CDC2 MRE1, CDK4 MRE1 and FEN1 MRE1 is significantly reduced upon co-transfection of HepG2 cells with miR-24 mimics (white) and not the control mimic (black). Mutations in the miR-24 pairing residues (mt) rescue luciferase expression (sequences and luciferase assays for all tested wt MREs for these genes in Suppl. Fig. 6,7). The CCNA2 MRE1 is not regulated. (F) However, miR-24 regulates a 181 nt region containing the CCNA2 MRE1 in the luciferase vector. Mutations in the binding residues of CCNA2 MRE1 within the extended sequence restore luciferase activity. Error bars in A, C and E represent mean ± SD from 3 independent experiments. **, p<0.01; *, p<0.05.

To verify further that MYC and E2F2 are direct targets of miR-24, we also looked at changes in expression of luciferase reporter genes 24 hr after transduction of HepG2 cells with miR-24 mimic. At this early time, thymidine uptake of HepG2 cells does not significantly change (Suppl. Fig. 4A), but ectopic miR-24 still suppresses luciferase reporters encoding MYC MRE3 or MRE6 or E2F2 MRE1 (Suppl. Fig. 4B). The identification of “seedless” E2F2 and MYC MREs confirms previous studies showing that MREs lacking a seed with good downstream complementarity can contribute to miRNA gene regulation (Didiano and Hobert, 2006, 2008; Vella et al., 2004).

Since “seedless” MREs contributed to the regulation of E2F2 and MYC by miR-24, we next investigated whether some of the E2F2 and MYC regulated genes, whose transcripts declined in response to miR-24, might also be direct miR-24 targets even though they might lack a predicted MRE. We selected 8 genes (AURKB, BRCA1, CCNA2, CHEK1, CDC2, CDK4, FEN1, PCNA) that play important roles in cell cycle progression and cloned their entire 3′UTRs into the luciferase reporter. The 3′UTR of 6 of 8 genes (AURKB, BRCA1, CCNA2, CDC2, CDK4, FEN1, but not CHEK1 or PCNA) was significantly repressed by miR-24, suggesting that these genes may be direct targets (Fig. 6C). To confirm that these genes are direct miR-24 targets, we next sought to identify the miR-24 MREs that regulate their expression. Among the 6 genes whose 3′UTR was specifically repressed by miR-24, BRCA1 is the only gene that is predicted by TargetScan. The BRCA1 3′UTR contains a nonconserved perfect 7-mer seed match sequence, which functions as a miR-24 MRE by luciferase assay (Fig. 6D,E). To identify potential miR-24 MREs in these E2F2 and MYC-regulated genes, we used the rna22 or PITA algorithms, which allow G:U wobbles or seed mismatches. These algorithms identified 1 candidate MRE for AURKB; 5 for BRCA1 (which included the TargetScan BRCA1 site); and 3 sites each for CCNA2, CDC2, CDK4 and FEN1 (Suppl. Fig. 6). miR-24 significantly repressed luciferase activity of one MRE for 5 of 6 of these reporter genes (AURKB MRE1, BRCA1 MRE5, CDC2 MRE1, CDK4 MRE1, FEN1 MRE1) (Fig. 6D,E; Suppl. Fig. 7). Although CCNA2 MRE1 appeared to be inactive, a longer fragment (181 nucleotides) from the CCNA2 3′UTR (that included only CCNA2 MRE1) significantly repressed luciferase expression in a miR-24-dependent manner when cloned into the luciferase vector 3′UTR (Fig. 6F). Point mutations that disrupt base-pairing between miR-24 and the 5 minimal MREs and the CCNA2 MRE within the extended sequence rescued luciferase expression, verifying that these MREs are regulated by miR-24 (Fig. 6D-F). Therefore we have identified, and verified by mutation, 7 seedless miR-24 MREs in genes important in cell cycle progression.

This work was supported in part by NIH AI070302 and a GSK-IDI Alliance grant (JL), by the NIA-IRP, NIH (KGB, MG), the Harry Oppenheimer Memorial Trust (WH), a GSK-IDI Alliance fellowship (FN) and the Harvard Center for AIDS Research (NY). We thank N. Dyson (Harvard Medical School) for the HA-E2F2 expression plasmid, Ray McGovern for programming support and Lieberman laboratory members for useful discussions.