Identification and characterization of miR 490 targets. (A) Schema delineating the workflow model for target identification delineates 2 distinct pathways based on mRNA degradation or translational control. Supplemental Tables 5 and 6 detail miRNA/mRNA pairs for either pathway. (B) Schema of DAAM1 mRNA (RefSeq accession no. NM_014992) with open-reading frame (ORF) and miR 490 binding sites is shown (top); topology of the DAAM1/miR 490 binding and calculated minimum free energy (MFE) are displayed as predicted using RNA hybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid). (C) Luciferase reporter assay quantification was completed 24 hours after transfection of HEK 293 cells using the 975-bp DAAM1 3′-UTR inserted downstream of Renilla luciferase (DAAM1) or cotransfected with a fixed concentration (10nM) of miR 490 3p or miR 490 5p chemical mimetics (or nontargeting Caenorhabditis elegans miR 239b stem-loop primer as control). Functional luciferase data from individual wells are expressed as the normalized mean ± SEM relative to DAAM1 expression (N = 6 wells/group representing aggregate data from 2 independent experiments). P values are calculated by paired t test.