An AGO2 adaptor expands the functional and evolutionary reach of microRNA targeting

Abstract Current models of microRNA (miRNA) silencing posit that RNA-sequence rules are sufficient for canonical targeting of mRNAs by Argonaute 2 (AGO2), the central protein of the miRNA-induced silencing complex (miRISC). Using chimeric eCLIP in CRISPR-edited LIMD1 +/+ , LIMD1 +/− , and LIMD1 −/− human small airway epithelial cells (hSAECs), we reveal a transcriptome-wide dependency on LIMD1, an AGO2 adaptor, for effective miRNA targeting and repression. In LIMD1-deficient cells, miRNA loading is uncoupled from productive targeting: despite increased AGO2–miRNA interactions, complexes engage fewer transcripts and sites, reducing occupancy and more than halving both the breadth and depth of targeting. We also observe altered AGO2 positional footprints across targets in LIMD1-deficient cells. LIMD1 dependence is most pronounced at defined RNA contexts: weak (GC-poor) seed pairings, interactions involving evolutionarily young miRNAs or sites that nonetheless form thermodynamically stable duplexes, with these losses particularly enriched in coding sequences of rapidly evolving C 2 H 2 -zinc-finger genes. Even within canonical seed repertoires of individual AGO2–miRNAs, LIMD1 is most critical at poorly conserved sites, indicating that LIMD1 broadens miRNA regulation beyond ancient, deeply conserved targets. In culture, LIMD1 deficiency de-represses oncogenic proteins that, in vivo , inversely correlate with LIMD1 levels in normal lung and adenocarcinoma, where LIMD1 is characteristically reduced, and whose dysregulation predicts poor survival. Thus, LIMD1 emerges as a key determinant of miRISC architecture, targeting, and potency, challenging RNA-centric models of miRNA function and exemplifying how adaptor proteins diversify post-transcriptional regulation. Graphical Abstract LIMD1 defines the scope of miRNA-mediated targeting and repression AGO2-chimeric eCLIP in CRISPR-edited human small airway epithelial cells (hSAECs) shows LIMD1 is required for productive AGO2–miRNA engagement transcriptome-wide. LIMD1 deficiency reduces the AGO2–miRNA:targetome. Each AGO2–miRNA binds fewer targets, with lower occupancy per site and per transcript and fewer global silencing events. LIMD1 dependence is strongest for GC-poor seed-sites, less conserved miRNAs and sites, and thermodynamically stronger duplexes. Dosage-dependent effects of LIMD1 deficiency are broadly observed. Target-mRNA decay and translational repression are reduced in LIMD1-deficient hSAECs, increasing protein output. In vivo , LIMD1 LOH–associated deficiency is prevalent and typically clonal in NSCLC. LIMD1 expression inversely correlates with oncogene levels in normal lung and adenocarcinoma, and target dysregulation predicts poor survival.