miR-1 complementary sites in 3′-UTR of repressed genes (A) The occurrence of the miR-1 seedmatch (CATTCC) per kb of 3′-UTR and coding sequence was calculated for the 12 miR-1 regulated genes, the remaining identified genes and for the entire 3′-UTR and coding sequence databases (Supplementary Table 1 online). P-values were calculated using a parametric bootstrap test comparing the observed value to a distribution of values obtained by repeated calculation miR-1 seedmatches per kb for 12 of 3′-UTRs or coding sequences randomly chosen from the set of identified genes (see Supplementary Figure 1). (B) Overlap between our data and the combined computational predictions of miR-1 targets (Supplementary Table 1 online). The P-value for the overlap was calculated with Fisher's exact test for count data. (C) Relative luciferase activities for reporter constructs containing the indicated 3′-UTR sequences (Supplementary Table 2 online). Error bars indicate the SD (n = 10). For each transfection firefly luciferase activity was normalized to renilla luciferase activity. For each construct the values are normalized to control miR transfection. P-values was calculated by comparing the ratio between the miR-1 and miR-control transfections to the same ratio for the empty vector with the Student's t-test (n = 18) and correcting for multiple testing with the Bonferroni correction. Standard deviations for the ratios were calculated using standard error propagation. P-values <0.05 are indicated. Data and calculations are included in Supplementary Table 2 online.

Identification of miR-1 targets with quantitative MS. (A) Schematic representation of the experimental set-up and the SILAC method. (B) Histogram of the 504 log2 transformed protein ratios determined in this study. The distribution is skewed to the left, but if the 20 most negative ratios are removed the resulting distribution (colored in red) is normal (Lilliefors ‘Kolmogorov–Smirnov’ normality test, n = 484, P = 0.1202). Comparing to this distribution 12 proteins (colored in blue) are repressed by miR-1 (P < 0.001, one-sided parametric bootstrap test) (Supplementary Table 1 online). (C) 398 of the protein ratios obtained in this study plotted against corresponding mRNA ratios (10) obtained 24 h after miR-1 transfection. (D) Venn diagram of genes regulated on the mRNA level by miR-1 after 12 and 24 h (10) and the miR-1 repressed set of proteins identified in this study. P-value for the overlap is calculated with Fisher's exact test for count data.