(A) Conservation of chimera-supported non-canonical sites detected in an analysis modeled after that of Grosswendt et al. (2014) but modified to control for background conservation. Plotted for the indicated miRNAs is the average conservation of chimera-supported non-canonical sites, as measured by branch-length score (BLS), compared to the average conservation of 100 equally sized cohorts of controls; error bars, standard deviation of cohort averages; **, p < 0.01; *, p < 0.05, one-sided Z test. We considered chimera-supported non-canonical sites that mapped within 3′ UTRs and contained a single mismatch to the 6 nt seed of the miRNA. This set of sites mirrored that analyzed previously (Grosswendt et al., 2014), and excluded offset 6mers, which as a class was already known to mediate repression and exhibit preferential conservation (Friedman et al., 2009). Cohorts of control sites were generated such that for each chimera-supported site, each control cohort contained a single example of the identical 6 nt motif that was present in the indicated region (either an AGO cluster or 3′ UTR) but not supported by chimeric reads. To control for local background conservation and thereby avoid treating sites within slowly evolving 3′ UTRs the same as those within rapidly evolving 3′ UTRs, we used the binning procedure developed for calculating PCT scores (Friedman et al., 2009); 3′ UTRs were partitioned into 10 conservation bins (based on the median BLS of the nucleotides of the human sequence), and control sites were randomly selected (with replacement) from 3′ UTRs in the same bin as the actual site. Control AGO clusters were collected as was done previously (Grosswendt et al., 2014), using genome-wide data downloaded from clipz.unibas.ch and derived from multiple AGO PAR-CLIP experiments performed in HEK293 cells (Kishore et al., 2011). The union of AGO clusters for all experiments was computed and filtered for overlap with Ensembl-annotated 3′ UTRs, using the ‘merge’ and ‘intersectBED’ utilities, respectively, found in BEDTools v2.20.1 (parameter ‘-s’) (Quinlan and Hall, 2010). (B) Attribution of the conservation signal to the confounding effects of conserved regions. Considered are 1443 non-canonical chimera-supported sites selected as in (A) but including sites of all miRNA families. For each chimera-supported site, a z score was generated using the distribution of BLSs for 100 control sites chosen as in panel (A) from either AGO clusters or 3′ UTRs, as indicated. Each z score reflected how the conservation of the actual site differed from that of its controls. Compared are cumulative distributions of the z scores for sites of broadly conserved miRNAs and those of less conserved miRNAs, using the previously defined sets of broadly and less conserved miRNAs (Friedman et al., 2009). If the chimera-supported non-canonical sites were preferentially conserved because of their function in mediating repression, then sites of broadly conserved miRNAs would be expected to have a right-shifted distribution compared to sites of less conserved miRNAs. However, no significant difference was discerned between each pair of z-score distributions. The remainder of this legend outlines the rationale for the analysis of this panel. One way to reconcile the conservation signal observed in panel (A) with our conclusion that a large majority if not all of these sites bind miRNA but do not mediate repression is to consider the potentially confounding biochemical properties of conserved regions, which are illustrated by the observation that artificial siRNAs preferentially target sites that are evolutionarily conserved over those that are not (Nielsen et al., 2007). Because these siRNAs are not natural (and do not share a seed with conserved miRNAs) the evolutionary conservation of these preferred sites could not have arisen because they function to mediate sRNA-guided repression. Instead, some other function of these 3′-UTR regions, such as greater accessibility to RNA-binding factors, must explain their preferential conservation and also endow them with properties that favor sRNA binding (Nielsen et al., 2007). To examine whether confounding properties of conserved 3′-UTR regions might similarly explain the elevated conservation of chimera-supported sites, we compared the z scores for sites bound by broadly conserved miRNAs (miRNAs in families conserved beyond mammals, as listed in TargetScan7) with those bound by less conserved miRNAs. MicroRNAs conserved among mammals but not more broadly were grouped with the less conserved miRNAs because canonical 6mer and 7mer sites to these miRNAs have no conservation signal above background, presumably because these miRNAs have not been present long enough for the number of preferentially conserved 6mer and 7mer sites to rise above the background (Friedman et al., 2009); we reasoned that the same would be true of non-canonical sites, to the extent that any are preferentially conserved. If the conservation signal observed in panel (A) were related to miRNA binding, we would have expected a difference between the scores for the sites of broadly and less conserved miRNAs. The lack of a significant difference supports the idea that chimera-supported non-canonical sites tend to be conserved for the same reason that functional sites to artificial siRNAs tend to be conserved.