Motifs with a high SLCS are enriched for miRNA target sequences. The distribution of non-zero SLCSs (black bars) from a 17-genome alignment and the distribution that resulted from an analysis of a randomly permuted genome are plotted. The distribution demonstrates that there are significantly more high-scoring motifs within coding regions compared with the distribution of scores from a randomized alignment (white bars). The highest-scoring sequence motifs (Inset) are enriched for miRNA target sequences. Results are filtered so that conserved motifs are removed if they are within 3 bp of a higher-scoring motif.

The microRNA let-7 targets the coding region of Dicer. (A) Dicer contains three highly conserved let-7 target sites. Alignments between the let-7 miRNA and the target sites are shown. The first two sites are conserved in 16 of 17 genomes, and the last is conserved in all 17 genomes. The coding sequence of Dicer is shown above let-7b. (B) HEK 293 cells were cotransfected with Dicer and with let-7b pre-miRNA or a pre-miRNA control. Several variants of the Dicer construct were used, from which various combinations of the let-7 sites had been abrogated by site-directed mutagenesis. (Upper) An immunoblot of Dicer levels and GAPDH levels. Wild-type Dicer is in lanes 1 and 2, fully mutated Dicer is in lanes 3 and 4, and each subsequent pair of lanes is marked to indicate which sites were intact (✓) and which were abrogated (×). Cellular lysates were analyzed by immunoblotting with antibodies to the FLAG epitope (upper band) or GAPDH (lower band). (Lower) The fold reduction in the presence versus absence of exogenous let-7 for each Dicer variant. Means (n = 4) and standard errors are plotted. Wild-type Dicer is significantly down-regulated by let-7 compared with the Dicer variant with all three target sites abrogated (two-tailed t test, P < 0.01). The Dicer variant with the first two target sites intact is also significantly down-regulated (P = 0.02). The Dicer variant with the first and third target sites intact is down-regulated with the next-highest magnitude, but the effect did not reach significance. Dicer variants with a single site intact are down-regulated less strongly than variants with two sites.

Highly conserved let-7 targets within coding regions are functional. Cultured human fibroblasts were transfected with pre-let-7b or a control RNA. Twenty-four hours after transfection, RNA was isolated, labeled, and hybridized to a whole-genome microarray. The fold change in expression between cells transfected with let-7b or the control was determined. The cumulative distribution of the fraction of genes down-regulated in the microarray data shows a significant difference between a random set of genes and genes with target sites in their 3′ UTR (P = 0.014, one-tailed Kolmogorov–Smirnov test), but not with genes with target sites in their coding region (P = 0.26). Genes with highly conserved target sites in their coding region, however, are significantly different from the set of random genes (P < 10⁻⁴), as are genes with highly conserved target sites in their 3′ UTRs (P < 10⁻³).

Recognition sites at various positions within a transcript have different patterns of miRNA binding. Sites complementary to the let-7 seed were extracted from 3′ UTRs (A), coding regions (B), and 5′ UTRs (C), along with additional upstream sequence, and computationally folded to a consensus let-7 miRNA (see Materials and Methods). The fraction of target–seed interactions in which a particular base pair was bound was determined for each position of the miRNA outside of the seed. The fraction bound was compared for highly conserved versus poorly conserved target seeds. Numbers indicate the number of genomes in which a seed was conserved; 12+ indicates indicates conservation in 12 or more genomes, 3− indicates conservation in three or fewer genomes. Statistical comparisons were performed by using the Fisher exact test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Different cutoffs for the number of genomes in the “conserved” and “nonconserved” groups were used for each search region, and target seeds of 6 bp were used for 5′ UTRs to ensure an adequate number of sequences on which to perform statistical analysis (Fig. S3 shows results for various conservation levels).