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. 2015 Jun 4;11(6):e1005245. doi: 10.1371/journal.pgen.1005245

Fig 6. Comparison of gain-of-function activities of miR-279 and miR-996.

Fig 6

(A) Luciferase sensor assays in S2 cells indicated that 3' UTRs of multiple miR-279 targets are all additionally responsive to miR-996. The control Hairless 3' UTR has no miR-279/996 seed match and was not repressed by these miRNAs. Error bars represent standard deviation from quadruplicate assays. (B) Northern confirmation of ectopic miR-279 and miR-996 in S2 cell experiments. pre = pre-miRNA hairpin, mature = mature miRNA product. Overexpressed miRNAs were calculated relative to endogenous mature miRNAs, normalized to 2S loading control. (C-D) Averaged activity profiles for control and miRNA overexpressing flies for 7 days in constant darkness since the second day after transferring from LD to DD. Some of these experiments utilized the amplifier driver tim-UAS-Gal4, as schematized in (E). (C) Overexpression of miR-279 by tim-Gal4 induced strong arrhythmia. (D) Ectopic expression of miR-996 by tim-Gal4 had no significant affect on circadian behavior, but further induction by tim-UAS-Gal4 led to a complete arrhythmia. n = ~32 for each genotype; the number of flies assayed for each genotype is indicated in Table 2. (F) Validation that higher levels of mature miR-996 are induced by tim-UAS-Gal4, compared to tim-Gal4. Overexpressed miRNAs were quantified as in (B) using Northern blotting, and normalized to 2S loading control. These tests also confirm that tim-Gal4>UAS-mir-996 flies effectively misexpressed miR-996, even though they lacked circadian defects.