mRNA isoform diversity complicates measurement of translational control by let-7. HeLa cells were transfected with anti-miRs, harvested 6–17 h later, and lysates fractionated by gradient centrifugation as in Figure 1. (A) 3′ UTR configurations of the 11 experimentally verified let-7 target mRNAs under study. Alternative polyadenylation sites (blue triangles) were identified from Refseq, EST, and the PACdb (Brockman et al. 2005) databases (see Supplemental Materials and Methods). Let-7 target sites (purple lines) were as predicted by Targetscan (Friedman et al. 2009) and other sources (MYC, Kong et al. 2008; GPR56, Selbach et al. 2008; HMGA2, Lee and Dutta 2007; IGF2BP1, Mayr and Bartel 2009). Also shown are binding sites for miRNAs expressed at the level of ≥10% that are seen for let-7 in HeLa cells (determined by small RNA-Seq; yellow lines). Binding sites for these miRNA were derived from published studies and Targetscan predictions (details in Supplemental Table S2). (B) Gradient distribution of let-7 target mRNAs after transfection anti-let-7 (gray squares) or control anti-miR (black circles). All qRT–PCR measurements used primers designed against coding regions. Profiles are the averages of four to nine independent experiments (bars represent standard error). An exception is KRAS, which is an average of three independent experiments (6–9 h after anti-miR transfection).
