Figure 1. Destabilization of reaper and hid reporter mRNAs by endogenous miRNAs.
(A) Schematic of reporter mRNAs analyzed; WT denotes mRNAs with wild type MREs and MT denotes mRNAs where the MREs were inactivated by mutation. FL is the firefly luciferase coding region and V5 is the V5 epitope tag. (B) Stability of reaper reporter mRNAs containing wild type (WT) or mutant (MT) MREs. Cells were treated with actinomycin D for the indicated times; total RNA was prepared and analyzed by Northern blotting using a probe to the firefly luciferase open reading frame. Rp49 denotes the mRNA encoding ribosomal protein L32, a highly stable mRNA, which served as an internal loading control. (C) Stability of hid reporter mRNAs containing wild type (WT) or mutant (MT) MREs. All procedures were exactly as in (B).
Figure 1—figure supplement 1. Sequences of 3’ UTRs of reaper and hid with wild type and mutant MREs highlighted.
(A) Reaper 3’ UTR. The wild type MRE is shown in red while the mutant MRE is shown in blue. (B) Hid 3’ UTR. Wild type MREs for bantam are shown in red while mutant MREs are shown in blue.
Figure 1—figure supplement 2. miRNA mimics complementary to mutant MREs destabilize reaper and hid reporter mRNAs.
For miRNA mimics, ds RNAs were ordered from Integrated DNA Technologies (IDT) according to their protocol for design of miRNAs. The mir2 mimic was synthesized to contain a seed sequence complementary to the altered nucleotides in the mutated reporter reaper reporter UTR. For the Bantam mimic, the seed was complementary to the new target sequence shown in supplemental Figure 1—figure supplement 1 present in all of the altered Bantam sites. The control dsRNA was a negative control recommended by IDT. For optimization of transfection conditions a double stranded siRNA of the same design was used to target the Firefly luciferase coding sequence. Titrations of dsRNA, plasmid and transfection reagent (Mirius Transit-X2) were performed to optimize expression and knockdown conditions as assayed by Luciferase readings in cells cotransfected with Firefly siRNA. For half life measurement, S2 cells were co-transfected with reporter and dsRNA in 10 cm plates at a starting density of 2X106/ml. 24 hr after transfection, cells were removed from plates, distributed on 6 well plates and 0.1 mM CuSO4 was added for an additional 16 hr at which point 5 µg/ml Actinomycin D was added, media was removed at the indicated times and cells were lysed with Trizol. Because of the low abundance of transfected reporter RNAs, the analysis of RNA half life was done by semi-quantitative reverse transcription PCR (Maroney et al., 2006). cDNA was synthesized from 0.5 µg total RNA using random primers and Superscript II. PCR reactions were first titrated for linearity using a series of cDNA dilutions and cycle numbers. Linear PCR reactions (25 cycles for Reaper and Hid reporters, 20 cycles for Rp49) were carried out in the presence of α32P dCTP and analyzed on non-denaturing 6% polyacrylamide gels.