Figure 1.
ARE-mediated mRNA decay is independent of Dicer in MEFs.
A) Total RNA was extracted from wt and dicer ko MEFs, and the expression of miR-16 was examined by Northern blot analysis. dicer ko MEFs show an accumulation of the precursor pre-miR-16, whereas mature miR-16 is detected only in the wt MEFs. U6 snRNA serves as a loading control. B) Degradation of the ARE-containing ier3 mRNA was examined in wt and dicer ko MEFs. Cells were treated with 5 µg/ml Actinomycin D and RNA was isolated at the indicated time points. Then, ier3 mRNA levels were measured by quantitative real-time PCR and normalized to nucleolin mRNA levels. Shown are average values ± SD from three biological repeats. C) ier3 mRNA half-lives were calculated from the decay curves, and plotted as average values ± SD in the graph on the right side.
Figure 2.
ARE mediated repression is independent of the mi/siRNA pathway in Drosophila.
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 Drosophila 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 tis-11. 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 (p<0.05, student's t-test, n = 4), C) left panel: response of the reporters (same as in B) to depletion of dcr-1 or dcr-2; right panel: response of the reporters (same as in B) to depletion of ago1 or ago2. D) Analysis of mRNA steady-state level changes upon depletion of tis-11, ago2 and dcr-2. Quantitative RT-PCR analysis was performed for the indicated mRNAs (cecA1 = endogenous ARE target, GFP-3xARE = stable ARE reporter, rp49 = ribosomal protein mRNA), normalized to the transcript levels of gapdh, and changes were calculated relative to control RNAi directed against DsRed. The asterisk (*) indicates a significant change relative to control RNAi (p<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.
Figure 3.
ARE mediated repression is independent of miR-289 activity.
A) Response of the GFP reporters (stable, polyclonal cell lines; miR-277 reporter: stable clonal cell line) to direct inhibition of miR-277 or miR-289; an antisense oligonucleotide directed against part of the firefly luciferase coding sequence served as control for normalization; values are the mean of two experiments. B) Response of the GFP reporters (same as in A) to overexpression of miR-277 or miR-289 (transient transfection); an unrelated plasmid (pUC18) served as control for normalization; values are the mean of two experiments. C) Northern Blot to test expression of miR-289; the same membrane was stripped and hybridized with the indicated probes.