Figure 1.

Method for detecting preferential conservation of miRNA sites. (A) Sites matching the miRNA seed region. All four canonical sites (colored) share six contiguous Watson–Crick matches to the miRNA seed (nucleotides 2–7); the offset 6mer contains six contiguous matches to nucleotides 3–8. (B) Phylogenetic conservation of individual sites. Each panel represents a miR-1 8mer site conserved to a branch length of 1.0. The 3′UTR of SLC35B4 falls into the fourth-most poorly conserved bin, which has a phylogeny with relatively long branch lengths (left), whereas the 3′UTR of SPRED1 falls into the most well-conserved bin, which has a phylogeny with shorter branch lengths (right). Branch segments connecting the species with sites are colored purple, with numbers indicating the lengths of the longer segments. The lengths of the segments connecting the species are summed to yield the branch-length score (equation). (C) Performance of controls matched for number of occurrences in human 3′UTRs, GC content, or expected conservation as calculated by a first-order Markov model. For each possible RNA 7mer, conservation rates (signal) and average conservation rates for 50 control 7mers (background) were calculated for all 3′UTRs at a branch-length cutoff of 1.0. Error bars indicate 25th and 75th percentiles. Because most 7mer motifs are not selectively maintained, well-performing controls should yield median signal-to-background ratios near 1.0, with low variability for every bin. (D) Nested site conservation. Because the seed-matched sites are interrelated, we subtract conserved instances of extended sites from those of shorter sites. This hypothetical site is an 8mer match to miR-1 in a human 3′UTR that is more broadly conserved as a 7mer-m8 than as an 8mer site, and as a 6mer than as a 7mer-m8 site. As a result of nested subtraction, our method considers this site an 8mer at low branch-length cutoffs but not a 6mer or 7mer. At moderate branch-length cutoffs, it switches to a conserved 7mer-m8 site, and at high branch-length cutoffs it switches to a conserved 6mer site but not a 7mer or 8mer. (E) Signal and background for the miR-1 8mer site. For each UTR bin 1 through 10, with bin 1 having the least conserved UTRs and bin 10 the most conserved, the number of miR-1 sites conserved at the indicated branch-length cutoff is plotted with estimated background (small plots). Results for all 10 bins were then combined to represent the aggregate signal and background for this site (large plot).