siRNA-directed mRNA repression is enhanced by local conservation and AU content. (A) Mean percent conservation at UTR positions within 100 bp 5′ (left) and 3′ (right) of conserved (red) or nonconserved (blue) extended seed matches to the set of conserved vertebrate miRNAs used in Lewis et al. (2005); overall UTR conservation was controlled for in the comparison of conserved and nonconserved seed matches. The average conservation differs significantly for the 100 bases 5′ and 3′ of the seed match (P < 10−200 by rank sum test for both). (B) Mean AU content (sets controlled for UTR AU content); average AU content is significantly different both 5′ and 3′ of the seed match (P < 10−18, P < 10−30), respectively. (C) Mean nLFC for three equal-sized mRNA sets binned by percent conserved positions (in HMRD) in the 50-nt region immediately 5′ (orange) or 3′ (purple) of siRNA seed matches (5′ region ends at position t10; 3′ region begins one base 3′ of position t1) for mRNAs containing single extended seed match to the relevant siRNA. Bars indicate standard error of the mean. Set size and mean percent conservation for each set are reported above and below each bar, respectively. P-values are for two-sided rank sum tests between the first and third bins. For both 5′ and 3′ conservation, the three bins have been sampled such that their distributions of overall UTR conservation, 5′ (or 3′) AU content, overall UTR AU content, seed match type, and initial expression level are not significantly different (P ≥ 0.05) (Supplemental Fig. S9). (D) Same as C, but with UTRs binned by AU content in the same 50-nt regions. Bins are sampled to control for UTR AU content, 5′ (or 3′) conservation, overall UTR conservation, seed match type, and initial expression level (NS = not significant at P-value cutoff 0.05).
