FIGURE 2:

The miR-185–binding region of mCry1-3′UTR acts as a cis element in translation repression. (A) miRNA target prediction algorithms (MIRanda, MIRBase, and TargetScan) were applied to screen for miRNAs with the potential to bind the 3′UTR of mCry1. (B) Predictions of mCry1-3′UTR (lower strand) and miR-185 (upper strand) hybrids were performed using RNAhybrid software (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid; Rehmsmeier et al., 2004). (C) Schematic representation of the reporter constructs. One or two copies of the miR-185 recognition element of mCry1-3′UTR (miR-185 MRE) were fused to the Renilla luciferase reporter gene. Firefly luciferase was used as a transfection control. (D) Luciferase activity was determined in NIH 3T3 cells transfected with RL (control), RL-2×185B, or RL-1×185B plasmids. The relative luciferase activity (ratio of RLUC/FLUC) was set to 100. Results shown are the mean ± SEM (n = 4; ***p < 0.0001).