Identification of 6 miRNAs targeting the GARP 3’ UTR in 293 cells.
A. 293 cells were cotransfected with a reporter plasmid and the indicated miRNA mimics (black regular: 20 miRNAs predicted to bind the GARP 3’ UTR and expressed in T cells; grey italic: negative controls). The reporter plasmid contains the GARP 3’ UTR cloned downstream of the Renilla luciferase gene, and a Firefly luciferase gene to control for transfection efficiency. Graphs indicate the ratio of Renilla to Firefly activities in cotransfected cells, normalized to the ratio in cells transfected with the plasmid alone (no miRNA). Data presented are the mean normalized ratios + SD measured in 3 to 12 independent experiments. * p < 0,0001 by comparison to control random miRNA for normalized ratios <1 (unpaired two-tailed Student’s t test). B. Schematic representation of the GARP 3’ UTR region, with predicted miRNA binding sites indicated by black boxes. The end of the truncated 1.7 kb 3’ UTR region cloned in a lentivirus used in Figure 9 is indicated by an arrow. C. Nucleotide sequences in black correspond to regions of the GARP 3’ UTR where the indicated miRNAs are predicted to bind (subscript numbers indicate positions relative to the first nucleotide after the STOP codon). miRNA sequences are shown in green, with seed regions underlined. Optimal alignments between the miRNA and the partial GARP 3’ UTR sequences were calculated with the mfold software . Red letters and red boxes indicate nucleotides in the GARP 3’ UTR that were substituted or deleted by targeted mutagenesis, respectively. D and E. 293 cells were cotransfected as in A, except that the reporter plasmid contained wild type (WT) or mutated (mut) forms of the GARP 3’ UTR, as indicated. Data presented are the normalized ratios of Renilla to Firefly activities (means of triplicates + SD) and are representative from 2 to 4 independent experiments.