Fig 3. The miRISC as well as the putative miR-15/16 target sites in the cyclin E1 3’UTR are functional.

A. Tethering of either Ago2 or the effector domain of GW182 represses translation of a reporter in mammalian cells as well as in Xenopus oocytes. Schematic representation of reporters used. mRNAs encoding lambda-N peptide with an HA-tag or an HA-tag only, fused to GW182 effector domain or Ago2 were either injected into oocytes for 24 h prior to injection of Renilla luciferase reporter mRNA containing 3’UTR Box B sites (Rluc-BoxB) or transfected into HeLa cells 24 h prior to transfection of plasmids encoding the reporter genes. An mRNA/plasmid encoding Firefly luciferase was co-injected/co-transfected as an internal control. After a 6 h incubation, cells were harvested and reporter protein expression was assessed. The experiment was repeated 3 times with similar results. B. Repression of a reporter mRNA tethered to the effector domain of GW182 is independent of a poly(A) tail. Schematic representation of reporters used. The experiment was performed in Xenopus oocytes as in A, using either non-adenylated (pA-) or in vitro polyadenylated (pA+) RNA. RNA was assessed from a pool of 50 injected oocytes. C. The two miR-15/16 sites co-operate in repressing translation of the cyclin E1 3’UTR. Luciferase reporters containing the wild-type cyclin E1 3’UTR (WT), mutations in the first miR target site (miR mut1), the second target site (miR mut2) or both (miR mut) were injected into stage VI oocytes. Renilla luciferase mRNA was co-injected as an internal control. Firefly luciferase levels are expressed as a ratio to Renilla internal control. The graph displays the results for a representative experiment. D. The miR-15/16 target sites are active in a degradation-independent manner. Luciferase reporters containing the wild-type cyclin E1 3’UTR (WT) or the cyclin E1 miR mut 3’UTR were injected into oocytes (see C for further details), in the presence of co-injected control or miR-15/16 LNAs, as indicated. * Student t-test P<0.01. RNA extracted from injected oocytes was subjected to reverse transcription and quantitative real-time PCR to assess RNA levels, which are expressed as a ratio of firefly to Renilla luciferase. The graph displays the results for a representative experiment.