Human Dicer hotspot mutation induces both loss and gain of miRNA function [RNA-seq]

The core miRNA biogenesis enzyme Dicer sustains recurrent mutations in cancer, which specifically compromise catalysis by its RNase IIIb domain. These are proposed to act as selective hypomorphs, since Dicer-RNase IIIb cleaves the 5p arms of pre-miRNA hairpins. However, the lack of knockin models has hindered a full understanding of the consequences of Dicer hotspot mutations for miRNA biogenesis. Here, we generated Dicer-KO and Dicer-S1344L (homozygous and hemizygous) hESCs; the latter is a non-catalytic mutation in RNase IIIa that impairs RNase IIIb activity. Consistent with our previous work, Dicer-KO is cell lethal in hESCs. However, in contrast to the expectation that Dicer hotspot mutations confer cell advantages, Dicer-S1344L knockin hESCs were subviable and also required a conditional genetic strategy for isolation. Small RNA sequencing using spike-in normalization reveals two broad trends: ablation of miRNA-5p species, and selective changes in miRNA-3p species. Curiously, we recognized a directional upregulation of miRNA-3p passenger strands, indicating a broad strand switch. Activity assays and Argonaute profiling of the transcriptome confirm that these confer increased repression capacity. Finally, we used in vitro assays to show 3p arm-nicked pre-miRNAs preferentially load miRNA-3p species into Argonaute, compared to corresponding miRNA/passenger duplexes. Overall, Dicer hotspot mutations have unexpected neomorphic consequences on miRNAs, including both loss- and gain-of-function.