Pathogenic DICER1 RNase IIIb Hotspot Mutation Induces 3p-miRNA Gain-of-Function via Argonaute Strand Switch [miR-seq]

Dicer is an essential enzyme in microRNA biogenesis. Mutations in the DICER1 gene are linked to various cancers, notably through the DICER1 syndrome. To investigate the impact of the pathogenic hotspot mutations in DICER1-associated tumors, we introduced a hotspot mutation into the endogenous Dicer1 locus of a mouse embryonic carcinoma cell line using CRISPR. Our findings not only confirm the loss of 5p-miRNAs, as previously reported, but also uncover an unexpected upregulation of specific 3p-miRNAs. These upregulated 3p-miRNAs, usually considered as passenger strands in the wild-type cells, are selectively loaded into the Argonaute protein in mutant cells based on their 5' end characteristics, resulting in a "strand-switch" phenomenon. Functional assays and transcriptome analyses demonstrate the passenger 3p-miRNAs’ activity. This study suggests that the Dicer hotspot mutation is not merely a loss-of-function mutation for 5p-miRNAs but also a gain-of-function mutation for passenger 3p-miRNA, potentially contributing to DICER1-associated tumorigenesis. To generate cell line isogenic to the DICER1 syndrome (P19 HM), P19 mouse embryonic carcinoma cell line was endogenously modified with CRISPR to harbor the biallelic loss-of-function mutation RNase IIIb hotspot mutation (mouse E1797G) on the Dicer1 gene. P19 HetKO (with only loss-of-function mutation on one allele) is included as a control. For Dicer overexpression experiments, wild-type of human E1813G Dicer was transfected in HEK293T Dicer KO cells. Sequencing was done with total RNA and AGO2-IP samples for each group with replicates. miR-seq was performed using the QIAseq miRNA Library Kit for all samples.