A Novel Anti-Apoptotic Role for Mir-290-295 Figure 1. Acute Dicer deletion and specific mir-295 cluster deletion show global target derepression signatures. A. PCR for confirmation of Dicer loss on acute deletion. Dicer floxed and floxed-deleted bands were amplified over a nine day time course to determine the point of maximal enrichment for Dicer cells. B. Northern blot for mature miRNAs following Dicer deletion. A DNA probe for the abundant miR-292 was assessed at 5, 7, and 9 days following Dicer deletion, with a probe for U6 to control for loading. C. Cumulative distribution functions (cdfs) of log2 fold change (LFC) in mRNA expression between Dcr WT and Dcr KO ES cells (top panel) and 295 WT and 295 KO ES cells (bottom panel) are plotted. Plots include conserved targets (red line), all predicted miR-295 targets (blue line), and control mRNAs (grey line). targets include ,3000 predicted TargetScan targets of the miR-295 cluster. Conserved targets contains the ,300 genes in the top 10% of targets ranked by TargetScan 5.1 branch length scores. The control mRNA set was selected to match the predicted targets in expression, 3’ UTR length and composition. Targets are derepressed in both Dcr KO as well as 295 KO mESCs (p#2.2e216 and p = 9.7e29 by rank sum test, respectively). Similarly, conserved targets are derepressed in both Dcr KO as well as 295 KO mESCs (p#2.2e216 and p = 3.1e214 by rank sum test, respectively). doi:10.1371/journal.pgen.1002054.g001 derepression observed with the luciferase reporter assay (Figure 3A). This observation indicates that the majority of miRNA repression likely occurs at the level of transcript stability. In support of the reporter assay, Dcr KO cells showed a comparable increase in Casp2 at the protein level, which could be partially rescued by transfection of either miR-295, miR-467a (which shares the same hexamer seed), or a Casp2 siRNA, but not by siRNAs against other unrelated targets (Figure 3B). Transfec- tion of miR-295 also strongly repressed an intact Casp2 reporter in these cells, but not a reporter in which the four target sites were mutated (Figure 3C). Combinatorial mutagenesis revealed that repression was not conferred equally by these four sites, as much of the repression was lost by mutation of the first two sites alone (Figure 3D). Taken together, these data suggest that direct miRNA-mediated repression of Casp2 leads to approximately 5- fold repression, making it one of the most potently repressed mir- 295 cluster targets identified to date. We additionally characterized the novel target Ei24, which has also been implicated in apoptosis. Originally identified as a direct p53 transcriptional target that binds Bcl2 [18,19], the Ei24 3’ UTR contains one 7mer miR-295 site. The 3’ UTR of Ei24 fused to a luciferase reporter conferred approximately 2-fold repression in Dcr WT cells relative to Dcr KO cells, an effect that could be restored following transfection of miR-295 (Figure 3C). Notably, repression was lost upon mutation of the seed site, confirming that Ei24 is a direct target. Mir-295 cluster miRNAs promote survival of ES cells during genotoxic stress Based on these repression data as well as the earlier informatic predictions, we tested whether mir-295 cluster miRNAs could modulate apoptosis in mESCs. The basal apoptosis rates of Dcr WT and KO ES cells in a 24 h period were compared by staining them with antibodies against cleaved Caspase 3 (Casp3) and then analyzing cells by flow cytometry. Under these conditions, only modest basal apoptotic rates were observed, with Dcr KO ES cells showing a slightly higher apoptosis rate than Dcr WT cells (Figure 4A, Figure S3A). Given that ESCs are highly sensitive to DNA damage [20] and both validated targets have been implicated in the DNA damage response, we hypothesized that the mir-295 cluster may be specifically protective in the context of genotoxic stress. To test this, we first examined the effect of exposing WT and Dcr KO cells to gamma irradiation or doxorubicin. Gamma irradiation induces DNA damage and activates ATM and p53, as does doxorubicin, a topoisomerase II inhibitor [21]. These signals lead to activation of the intrinsic apoptosis pathway and result in the cleavage of Casp3 [22]. We were able to confirm this cleavage product by Western blot in our cell culture system, as well as cleavage of Nanog, a previously reported Casp3 target [23] (Figure S5A). We also observed a decrease in Casp2 levels and the appearance of the previously described 35kD cleavage product [24], confirming its activation in our system (Figure S5B). Because this band was specific to DNA PLoS Genetics | www.plosgenetics.org 3 May 2011 | Volume 7 | Issue 5 | e1002054