Fig. 9: Evolutionary perspective on the regulatory impact of RBPs on miRNA targeting.

a, Fraction of miRNA target sites (MTSs) that have RBP-binding sites (RBSs) in their close proximity. 7, 8mer MTSs of miRNA families conserved across the vertebrates were collected⁴⁷. The fraction of MTSs including ≥1 RBSs within 50 or 100 nt flanking regions was calculated using the binding information of RBPs profiled in HepG2 (top left). Based on the calculated fractions for subsampled RBPs (solid dots), we fitted a regression curve (solid line) to extrapolate the analysis to a larger number of considered RBPs (dashed line). The same analysis was repeated for conserved (PhyloP⁶² > 0.0, top middle) and highly conserved MTSs (PhyloP > 2.0, top right). Similar analyses were performed for RBPs profiled in K562 (bottom). b Co-occurrence of evolutionarily conserved MTSs and RBSs in various human tissues. By utilizing transcriptome data of 54 tissues⁴⁸, highly expressed RBPs and miRNAs were collected for each tissue. For each conserved miRNA highly expressed in a given cell, 7, 8mer MTSs were collected and RBP-binding signals near the MTS were measured within 50 nt flanking regions. The magnitudes of RBP-binding signal were compared between the chosen MTSs and their control MTSs by using two-sided Wilcoxon’s rank-sum test after correction of confounding features (Supplementary Fig. 8c). Statistical significances after multiple test correction via the false discovery rate are displayed. Upward and downward directions of the y-axis indicate co-occurrence and mutually exclusive occurrence between MTSs and RBSs, respectively. c Proposed model of MT that takes >1,500 RBPs into account. For an MTS readily accessible to AGO, other RBPs would not affect MT efficacy (top row) and such cases are well explained by the currently accepted model of a ternary interplay between AGO, miRNA, and mRNA target. However, as our analysis indicated that in a vast majority of cases ≥1 RBSs are located close to the MTS, our proposed model that additionally takes RBSs of >1,500 3′UTR-binding RBPs into consideration would better explain MT (bottom four rows).