ARE mediated repression is independent of the mi/siRNA pathway in <i>Drosophila</i>.

<p>A) Schematic representation of the reporter constructs employed in our analysis; the sequence of one artificial ARE element is indicated 5′ to 3′ (modeled after the interferon-γ element). Bottom panel: Analysis of reporter-mRNA decay. Transiently transfected <i>Drosophila</i> S2-cells were treated with α-amanitin at 5 µ g/ml for 0, 1 and 6 hours. Introduction of the TNF-α-3′UTR sequence destabilizes the reporter mRNA. B) Response of the GFP reporters (stable, polyclonal cell lines) to depletion of the canonical AU-rich element binding factor <i>tis-11</i>. GFP fluorescence was measured by flow cytometry and normalized to control knock-down (dsRNA directed against DsRed); values are mean ± SD (n = 3). The changes were significant for 2xARE, 3xARE, IL-6 3′-UTR and TNF-α 3′-UTR when compared to the TNF-αΔARE control (<i>p</i><0.05, student's t-test, n = 4), C) left panel: response of the reporters (same as in B) to depletion of <i>dcr-1</i> or <i>dcr-2</i>; right panel: response of the reporters (same as in B) to depletion of <i>ago1</i> or <i>ago2</i>. D) Analysis of mRNA steady-state level changes upon depletion of <i>tis-11</i>, <i>ago2</i> and <i>dcr-2</i>. Quantitative RT-PCR analysis was performed for the indicated mRNAs (<i>cecA1</i> = endogenous ARE target, GFP-3xARE = stable ARE reporter, <i>rp49</i> = ribosomal protein mRNA), normalized to the transcript levels of <i>gapdh</i>, and changes were calculated relative to control RNAi directed against DsRed. The asterisk (*) indicates a significant change relative to control RNAi (<i>p</i><0.03, Wilcoxon's rank sum test, n = 6). E) Experiment analogous to B) and C) using transient transfection of the reporter constructs rather than stably expressing cells.