Mouse X-linked microRNA cluster regulates the meiotic checkpoint and <i>Prdm9</i> -driven hybrid sterility in a copy number–dependent manner

One of the reproductive barriers between diverging populations during formation of a new species is the sterility of their hybrids. The <i>Prdm9-</i>driven hybrid male sterility of <i>Mus musculus musculus</i> × <i>Mus musculus domesticus</i> hybrids depends on the interaction between PRDM9, a histone methyltransferase that determines the positions of meiotic recombination hotspots, and an as yet unknown X-linked genetic factor within the Hybrid sterility X2 (<i>Hstx2</i>) locus. Here, we report that the <i>Mir465</i> microRNA (miRNA) gene cluster is the predicted <i>Hstx2</i> hybrid sterility factor. We show that removal of the <i>Mir465</i> genes restores the fertility of sterile hybrids and improves meiotic synapsis of homologous chromosomes. <i>Mir465</i> knockout also restores spermatogenesis in sterile chromosomal translocation carriers, demonstrating that <i>Mir465</i> acts as a meiotic checkpoint that can be activated independently of <i>Prdm9</i> intersubspecific incompatibility. Furthermore, the <i>Mir465</i> knockout increases the global recombination rate in hybrids and in parental <i>Mus m. domesticus</i> mice. This demonstrates that <i>Mir465</i> is responsible for the phenotypes of the two overlapping genetic loci, the <i>Hstx2</i> engaged in fertility of hybrids and the Meiotic recombination 1 (<i>Meir1</i>) controlling the recombination rate. The finding of enlarged <i>Mir465</i> clusters in all European <i>Mus m. musculus</i> samples tested and the identification of differentially expressed targets suggest that the reproductive barrier between the two subspecies is sensitive to copy number variation of <i>Mir465</i> genes. Together, the underdominant interaction between <i>Prdm9</i> and <i>Mir465</i> provides a rare example of Dobzhansky-Muller incompatibility in hybrids of closely related species, making it accessible for further analysis at the molecular level.