Target Prediction and Luciferase Assays

Sequence-based miRNA target-prediction databases (e.g., microT-CDS,40 http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=microT_CDS/index; TargetScan,41 http://www.targetscan.org/) and coexpression-based tools for systematic miRNA target recognition (e.g., Co-expression Meta-analysis of miRNA Targets [CoMeTa];42 http://cometa.tigem.it/) were used to annotate miR-204-predicted targets.

Luciferase assays were performed as described.9 To test the direct regulation of the 3′ UTR of target genes by miR-204, the 3′ UTR-bearing vector was cotransfected with a synthetic miR-204 mimic (Dharmacon, Lafayette, CO, USA). Appropriate control vectors, in which the sequence of the candidate miRNA binding site at the 3′ UTR (i.e., predicted to be recognized by the miR-204 seed region) was mutagenized to abolish binding, were used in parallel.