MiRNAs regulate the expression of hepatic genes involved in pharmacokinetics and pharmacodynamics. Genetic variants affecting miRNA binding (mirSNPs) have been associated with altered drug response, but previously used methods to identify miRNA binding sites and functional mirSNPs in pharmacogenes are indirect and limited by low throughput. We utilized the high‐throughput chimeric‐eCLIP assay to directly map thousands of miRNA‐mRNA interactions and define the miRNA binding sites in primary hepatocytes. We then used the high‐throughput PASSPORT‐seq assay to functionally test 262 potential mirSNPs with coordinates overlapping the identified miRNA binding sites. Using chimeric‐eCLIP, we identified a network of 448 miRNAs that collectively target 11,263 unique genes in primary hepatocytes pooled from 100 donors. Our data provide an extensive map of miRNA binding of each gene, including pharmacogenes, expressed in primary hepatocytes. For example, we identified the hsa‐mir‐27b‐DPYD interaction at a previously validated binding site. A second example is our identification of 19 unique miRNAs that bind to CYP2B6 across 20 putative binding sites on the transcript. Using PASSPORT‐seq, we then identified 24 mirSNPs that functionally impacted reporter mRNA levels. To our knowledge, this is the most comprehensive identification of miRNA binding sites in pharmacogenes. Combining chimeric‐eCLIP with PASSPORT‐seq successfully identified functional mirSNPs in pharmacogenes that may affect transcript levels through altered miRNA binding. These results provide additional insights into potential mechanisms contributing to interindividual variability in drug response.

MicroRNAs (miRNAs) regulate protein-coding gene expression primarily through cognitive binding sites in the 3' untranslated regions (3′ UTRs). Seed sites are sequences in messenger RNAs (mRNAs) that form perfect Watson-Crick base-paring with a miRNA's seed region, which can effectively reduce mRNA abundance and/or repress protein translation. Some seedless sites, which do no form perfect seed-pairing with a miRNA, can also lead to target repression, often with lower efficacy. Here we report the surprising finding that when seedless sites and seed sites are co-present in the same 3'UTR, seedless sites attenuate strong-seedsite-mediated target suppression, independent of 3′ UTR length. This attenuation effect is detectable in >70% of transcriptomic datasets examined, in which specific miRNAs are experimentally increased or decreased. The attenuation effect is confirmed by 3'UTR reporter assays and mediated through base-pairing between miRNA and seedless sites. Furthermore, this seedless-site-based attenuation effect could affect seed sites of the same miRNA or another miRNA, thus partially explaining the variability in target suppression and miRNAmediated gene upregulation. Our findings reveal an unexpected principle of miRNA-mediated gene regulation, and could impact the understanding of many miRNA-regulated biological processes.