The AGO2 adaptor LIMD1 expands the functional and evolutionary reach of microRNA targeting

MicroRNA (miRNA) silencing is classically ascribed to RNA-sequence rules that guide Argonaute 2 (AGO2) targeting. Using chimeric eCLIP and complementary analyses in CRISPR-edited human lung epithelial cells, we show that efficient miRNA targeting also depends on the AGO2 adaptor-scaffold LIMD1. In LIMD1-deficient cells, AGO2 binds more miRNAs, but each AGO2-miRNA engages fewer transcripts and sites, reducing occupancy and halving both the breadth and depth of targeting. LIMD1 dependence is most pronounced for poorly conserved, weakly seed-paired sites that nonetheless form stable duplexes. LIMD1 deficiency alters AGO2 footprints and derepresses oncogenic targets inversely correlated with LIMD1 expression in lung adenocarcinoma. Thus, LIMD1 modifies the outcome of sequence-defined interactions that would otherwise be infrequent, unstable, or unproductive, revealing an adaptor-governed layer of posttranscriptional regulation beyond RNA-sequence rules.