Human Dicer1 hotspot mutation induces both loss and gain of miRNA function

The core miRNA biogenesis enzyme Dicer1 sustains recurrent mutations in cancer that compromise its RNase IIIb domain, which cleaves 5p arms of pre-miRNA hairpins. However, the lack of knockin models has limited fuller understanding. Here, we generated Dicer1-KO and Dicer1-S1344L (homozygous and hemizygous) human ESCs; the latter is a non-catalytic mutation in RNase IIIa that impairs RNase IIIb activity. Dicer1 knockouts lack canonical miRNAs, while S1344L induces two trends: ablation of miRNA-5p strands, and selective changes in miRNA-3p strands. Curiously, we recognized directional upregulation of miRNA-3p passenger strands, indicating a broad strand switch. We used multiple in vitro assays to show 3p arm-nicked pre-miRNAs preferentially load miRNA-3p species into Argonaute, compared to corresponding duplexes. Moreover, activity assays, RNA-seq data, and Argonaute-mRNA profiling, confirm that these confer increased repression capacity. These data expand the molecular consequences of Dicer1 hotspot mutations in cancer.