This decrease in sensitivity compared to supplementing additional let-7 is most likely due to reaching the level of endogenous let-7 levels in HEK 293 cells. reporter system quantifies cellular levels of the tumor suppressor miRNA let-7 in real-time in single Human embryonic kidney 293 (HEK 293) cells. Our data shows that the MiRAR can be applied to detect changes in miRNA levels upon disruption of miRNA degradation pathways. We further show that the reporter could be adapted to monitor another disease-relevant miRNA, miR-122. With trivial modifications, this approach could be applied across the miRNome for quantification of many specific miRNA in cell cultures, tissues, or transgenic animal models. and regulates the G2/M checkpoint in cell cycling and contains binding sites for let-7 JNJ 1661010 miRNA in its 3-UTR [24]. Let-7 also regulates apoptosis via let-7 binding sites in the 3-UTR of expression, let-7 allows cells to escape apoptotic effector caspases [17]. gene to a GFP reporter, allowing for a direct readout of let-7 activity in vivo, thus JNJ 1661010 generating a miRNA activity reporter (MiRAR). We further show that this MiRAR reporter system can be adapted as a reporter for other miRNAs. Our proof using principle experiments shows that genetically encoded sensors of miRNA activity will be highly useful tools in investigating the biological role of RNA-regulating enzymes in vivo. 2. Materials and Methods 2.1. Genomic DNA Extraction Genomic DNA was prepared as a template for the amplification of 3-UTRs as described previously [33]. Briefly, human embryonic kidney 293 (HEK 293) cells were grown to confluency in Dulbeccos modified eagle medium (DMEM) from Gibco (Thermo Fisher Scientific, Waltham, MA, USA) and 10% fetal bovine serum (FBS). Cells from half of a 150 mm plate (~107 cells) were resuspended in 2.4 mL lysis buffer (0.6% sodium dodecyl sulfate (SDS), 10 mM ethylenediaminetetraacetic acid (EDTA), 10 mM Tris-HCl pH 8.0) and 25 units RNase I (NEB M0243S) and incubated at 37 C for 1 h. This was followed by the addition of 240 L 5 M NaCl (6 mmol) and 1 h of incubation on ice. The solution was centrifuged at 10,000 for 30 min at 4 C and the DNA was extracted from the supernatant via phenol chloroform extraction. The extracted DNA was stored at ?20 C until further use. 2.2. Reporter Gene JNJ 1661010 Construct for Let-7 We generated a reporter system to link miRNA content in the cell to GFP fluorescence. GFP was cloned into pcDNA3.1. The 3-UTR of human wildtype was amplified from pRL KRas 3-UTR (plasmid 14804, Addgene, Cambridge, MA, USA) and mutant KRas 3-UTR from pRL KRas 3-UTR (plasmid 14805 Addgene) [34] using primers KRasfor (5-TCTGGGTGTTGATGATGCCTTC-3) and KRasrev (5-CCTGGTAATGATTTAAATGTAGTTATAGAAATAAATAATATG-3). The resulting PCR product was cloned downstream of GFP into pcDNA3.1-GFP using gene into pcDNA3.1 to generate a reporter system where GFP fluorescence is responsive to changes in let-7 concentration in the cell (Figure 1a). Cells transfected with the reporter construct pMiRAR-let-7 expressed GFP (Figure 2a,c), indicating that endogenous let-7 levels do not entirely silence expression. Background fluorescence of cells without miRAR (1858 44 RFU (relative fluorescence units)) was subtracted from the fluorescence intensities. To evaluate the responsiveness of GFP production, we co-transfected the reporter pMiRAR-let-7 with 80 pMlet-7 miRNA or separately with anti-miR RNA complementary to let-7 (anti-let-7). Supplementing cells with exogeneous let-7 effectively inhibited GFP translation, reducing fluorescence by more than 3-fold (Figure 2a,c). In contrast, supplementing cells with anti-let-7, which binds to and de-activates cellular let-7, led to a marked decrease in active let-7 molecules in the cell as reported by a 1.3-fold increase in GFP production and fluorescence (Figure 2a,c). Open in a separate window Figure 1 Schematic of the microRNA (miRNA) activity reporter (MiRAR) for let-7 levels in vivo. The KRas 3-UTR was fused downstream of green fluorescence protein (GFP) to allow quantification of cellular let-7 levels. (a) Schematic of pMiRAR-let-7 construct; (b) miRNA degradative enzymes Lin28 and Tut4 collaborate to mark let-7 miRNA for degradation by the exonuclease Dis3L2. The RNA binding protein Lin28 recruits Tut4 to polyuridylate Gata3 miRNA and pre-miRNAs, leading to degradation by the U-specific exonuclease Dis3L2. Lowered miRNA levels lead JNJ 1661010 to an increase in GFP translation and fluorescence. KRas: Kirsten rat sarcoma viral oncogene homolog, UTR: untranslated region. Open in a separate window Figure 2 The MiRAR in live cells. Fluorescence intensity measurements and cell images for different treatments of MiRAR-transfected cells. Human embryonic kidney 293 (HEK 293) cells were grown to confluency, transfected with the MiRAR containing the KRas-3-UTR, and treated as outlined. (a) Fluorescence intensities validate miRAR-let-7 as a miRNA reporter. Background fluorescence of untreated cells was subtracted from the experiments. Error bars are based on at least three biological.
