A high-resolution map of functional miR-181 target sites in the thymus reveals the role of coding sequence targeting and alternative seed binding

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNA, resulting in translational inhibition and/or mRNA degradation. Efficient targeting depends on a combination of biochemically established targeting rules and contextual factors of a given miRNA and cell type. Here, we generated a high-resolution map of miR-181 MREs to define the targeting rules of miRNA miR-181a/b-1 in murine T-cell development. We uncovered a broad array of novel functional targets of miR-181 and demonstrated that miR-181 acts predominantly through RNA destabilization. High-resolution analysis validated the dominance of seed matches. Critically, we also discovered a unique alternative seed match for miR-181 and identified a distinct group of targets controlled by translational inhibition. In conclusion, deep profiling of miRNA MREs in primary cells is critical to expand physiologically relevant miRNA targetomes and helps to establish context-dependent miRNA targeting rules. Overall design: Ribosome footprinting and RNAseq were performed with mouse thymocytes isolated from 3 WT and 3 miR-181a/b-1 KO mice.